Localization of nitric oxide synthase in the brain of the frog,Xenopus laevis

Nitric oxide synthase was localized in the brain of the South african clawed frog by NADPH diaphorase histochemistry and immunohistochemistry. All structures stained by the antiserum also displayed NADPH diaphorase activity. The fiber bundles of the terminal nerve, however, were positive for NADPH diaphorase but were not immunoreactive. In the forebrain, neurons expressing nitric oxide synthase were concentrated to the pallium, striatum, nucleus accumbens and anterior entopeduncular nucleus. Strongly stained neurons in the diencephalon were detected in the lateral thalamus, the tuberculum posterior and in the ventral hypothalamus. In the mesencephalon, the tectum and the magnocellular nucleus of the torus semicircularis contained many positive cells. Farther caudally, intensely stained neurons were abundant in an area corresponding to the anuran locus coeruleus, in the descending nucleus of the trigeminus and the inferior reticular nucleus. In the cerebellum, Purkinje cells were weakly stained. In summary, the expression pattern of nitric oxide synthase in the anuran brain reveals similarities to that of other vertebrates. The strongly positive cell group in the locus coeruleus may correspond to cholinergic cell groups in the mesopontine area in mammals.     

Distribution and synaptic relations of NOS neurons in the dorsal raphe nucleus: A comparison to 5-HT neurons

Anti-nitric oxide synthase antibody was used to study the distribution, cytowchhecture, and synaptic relations of nitric oxide synthase-like immunoreactive neurons in the whole rostral-caudel length of the dorsal raphe nucleus of the rat and compared them with serotonergic neurons. Results showed that the distribution of the nitric oxide synthase in the dorsal raphe nucleus was similar to that of the serotonergic neurons at the rostral part of the dorsal raphe nucleus, including the mediodorsal and the medioventral cell groups, and changed at the middle and caudal parts of the dorsal raphe nucleus. The cytoarchitecture of the nitric oxide synthase-like immunoreactive neurons in the medioventrai cell group of the dorsal raphe nucleus was similar to that of the serotonergic neurons. Similar to the serotonergic neurons there, nitric oxide synthase-like immunoroactive neurons also received synapses from axon terminals that contained round, or flattened vesicles, or both kinds. Different to the serotonergic neurons, the few nitric oxide synthase-like immunoroactive axon terminals that were in this area formed synapses.     

Termination patterns of serotoninergic medullary raphespinal fibers in the rat lumbar spinal cord: an anterograde immunohistochemical study

Electrical and chemical stimulation given in the ventral medullary raphe nuclei inhibits spinal nociceptive reflexes and spinal nociceptive transmission; serotoninergic receptors have been demonstrated to partially mediate that inhibition. In the present study, the termination patterns of raphespinal fibers in the rat lumbar spinal cord demonstrating serotonin-like immunoreactivity were examined by using the anterograde tracer Phaseolus vulgaris leucoagglutinin (PHA-L) in combination with immunohistochemistry. Fibers and terminations from the ventral medullary raphe nuclei (raphe magnus and raphe pallidus) demonstrating both PHA-L- and serotonin-like immunoreactivity were identified in all laminae of the dorsal horn and the ventral horn. Networks of large fibers, characterized by large boutons, and which did not demonstrate serotonin-like immunoreactivity, were identified in deeper laminae of the dorsal horn. The heterogeneous morphology of raphespinal fibers identified in the dorsal horn suggests that these fibers also may be heterogeneous in neurochemistry and function. Medial medullary sites outside the raphe nuclei were found to innervate the ventral horn and all laminae of the dorsal horn, with the exception of lamina I. Descending fibers and terminations also demonstrating serotonin-like immunoreactivity were identified in deep laminae (III, IV, V, VI) of the dorsal horn and in the ventral horn. Similarly, large fiber networks were identified which did not demonstrate serotonin-like immunoreactivity.     

Origins and terminations of bulbospinal axons that contain serotonin and either enkephalin or substance-P in the North American opossum.

We have shown previously that some enkephalin, substance-P, and serotoninergic neurons in the medullary raphe and adjacent reticular formation project to the spinal cord in the opossum. In the present study we have combined the retrograde transport of True Blue and immunofluorescence histochemistry to determine whether methionine enkephalin or substance-P containing bulbospinal neurons are serotoninergic. Furthermore, we have used the same immunofluorescence protocol to determine whether spinal axons contain the same substances. Neurons that immunostained for both enkephalin and serotonin were observed in many brainstem nuclei. However, those that projected to the spinal cord were limited to the nuclei raphe magnus and obscurus, and the ventral part of nucleus reticularis gigantocellularis, pars ventralis. Neurons that immunostained for both substance P and serotonin were fewer in number, but some of the ones in the above nuclei and within the nucleus raphe pallidus, projected to the spinal cord. Spinal axons exhibiting both enkephalin- and serotonin-like immunoreactivity were observed in the superficial laminae of the dorsal horn, lamina X, and the intermediolateral cell column, whereas those showing both substance-P and serotonin-like immunoreactivity were seen primarily in lamina X, the intermediolateral cell column, and the ventral horn. Some of the axons in the ventral horn were in close apposition to presumed motoneurons. Comparison of the above results with those obtained from previous studies of bulbospinal projections has allowed us to infer the origins of axons that innervate different spinal targets.     

Serotoninergic neurons in the brainstem expressing FOS protein after orofacial noxious stimulation: an immunocytochemical double-labeling study

Employing an immunocytochemical double-labeling technique, we investigated the co-localization of FOS protein, the expression product of c-fos proto-oncogene induced by orofacial noxious stimulation, and serotonin in the rat brainstem. About 3.2%-25.6% of serotonin-like immunoreactive neurons and 6.2%-46.7% of FOS-like immunoreactive neurons in the raphe nuclei, reticular formation and ventrolateral subdivision of the midbrain periaqueductal gray exhibit FOS-like immunoreactivity and serotonin-like immunoreactivity, respectively. The present results provide further morphological evidence for the involvement of serotoninergic neurons in modulating the transmission of noxious information.     

Pedunculopontine nucleus in the squirrel monkey: Distribution of cholinergic and monoaminergic neurons in the mesopontine tegmentum with evidence for the presence of glutamate in cholinergic neurons

The topographical relationships between cholinergic neurons, identified by their immuno-reactivity for choline acetyltransferase (ChAT) or their staining for β-nicotinamide ademine dinucleotide phosphate (NADPH)-diaphorase, and dopaminergic, serotoninergic, Nonadrenergic, and glutamatergic neurons that occur in the mesopontine tegmentum, were studied in the squirrel monkey (Saimiri sciureus). The ChAT-positive neurons in the pedunculopontine nucleus (PPN) form two distinct subpopulations, one that corresponds to PPN pars compacta(PPNc) and the other to PPN pars dissipata (PPNd). The ChAT-positive neurons in PPNc are clustered along the dorsolateral border of the superior cerebellar peduncle (SP) at trochlear nucleus levels, whereas those in PPNd are scattered along the SP from midmesencephalic to midpontine levels. At levels caudal toe the trochlear nucleus, ChAT-positive neurons corresponding to the laterodorsal tegmental nucleus (LDT) lie within the periaqueductal gray and extend caudally as far as locus coeruleus levels. All ChAT-positive neurons in PPN and LDT stain for NADPH-diaphorase; the majority of large neurons in PPN and LDT are cholinergic, but some large neurons devoid of NADPH-diaphorase also occurnin these nuclei. Cholinergic neurons in the mesopontine tegmentum form clusters that are largely segregated from raphe serotonin immunoreactive neurons, as well as from nigral dopaminergic and coeruleal noradrenergic neurons, as revealed by tyrosine hydroxylase immunohistochemistry. Nevertheless, dendrites of cholinergic and noradrenergic neurons are clolinergic and noradrenergic neurons are closely intermingled, suggesting the possibility of dendrodendritic contacts. In addition, numerous large and medium-sized glutamate-immunoreactive neurons are intermingled among cholinergic neurons in PPN. Furthermore, at trochlear nucleus levels, about 40% of cholinergic neurons display glutamate immunoreactivity, whereas other neurons express glutamate or ChAT immunoreactivity only. This study demonstrates that (1) cholinergic neurons remain largely segregated from monoaminergic neurons throughout the mesopontine tegmentum and (2) PPN contains cholinergic and glutamatergic neurons as well as neurons coexpressing ChAT and Glutamate in primates. © 1994 Wiley-Liss, Inc.     

The distribution of neuronal nitric oxide synthase in the nucleus tractus solitarii of the squirrel monkey

The distribution of neuronal nitric oxide synthase (nNOS) containing neurons and fibers in subnuclei of the nucleus tractus solitarii (NTS) in the squirrel monkey, Saimuri sciureus, was investigated by nNOS immunohistochemistry and nicotinamide adenine dinucleotide phosphate (NADPH)-diaphorase histochemistry. Generally, the staining pattern of nNOS and NADPH-diaphorase in the NTS was similar. A high density of neurons and fibers exhibiting both nNOS immunoreactivity and NADPH-diaphorase reactivity was present in the central, medial, intermediate, and dorsolateral subnuclei of the NTS. A moderate density of neurons and fibers that stained for both nNOS and NADPH-diaphorase was noted in the interstitial and ventromedial subnuclei. The gelatinosus and commissural subnuclei contained a low density of neurons and fibers exhibiting nNOS immunoreactivity and NADPH-diaphorase staining. The dorsal motor nucleus of vagus contained a high density of nNOS immunopositive and NADPH-diaphorase containing neurons and fibers at the rostral level, but contained a moderate density of positive fibers and very few positive neurons at the intermediate, subpostremal and commissural NTS levels. Incongruence was noted, however, between nNOS immunostaining and NADPH-diaphorase staining in blood vessels in the brainstem. Capillaries and small vessels exhibited strong staining for NADPH-diaphorase but no nNOS immunoreactivity. In summary, this work substantiates the presence of nNOS in subnuclei of the monkey NTS and is consistent with a role for NO(.) in neurotransmission in primate NTS.     

Coexistence of NADPH diaphorase with GABA,glycine, and acetylcholine in rat spinal cord

The enzyme NADPH diaphorase is present in many spinal neurons, and is thought to correspond to nitric oxide synthase. In order to determine which types of neuron in the spinal cord contain this enzyme, we have carried out a combined enzyme histochemical and immunocytochemical study with antibodies to GABA, glycine, and choline acetyltransferase. Two hundred and twenty-four NADPH diaphorase-positive neurons in midlumbar spinal cord from four rats were tested for GABA- and glycine-like immunoreactivity. The majority of these neurons (207/224) were GABA-immunoreactive and 139 were also glycine-immunoreactive. NADPH diaphorase-positive neurons in laminae I and II generally showed both types of immunoreactivity, while those in deeper laminae of the dorsal horn and around the central canal either showed both types or else were only GABA-immunoreactive. Since GABA and acetylcholine are thought to coexist in spinal neurons, NADPH diaphorase staining was combined with immunostaining for choline acetyltransferase. Immunoreactive neurons in laminae III and IV were all NADPH diaphorase-positive, while only some of those around the central canal and in the deeper laminae of the dorsal horn were positive. Choline acetyltransferase-immunoreactive neurons in the intermediolateral cell column (presumed sympathetic preganglionic neurons) were often NADPH diaphorase-positive, whereas those in the ventral horn (presumed motorneurons) were not. NADPH diaphorase-positive cells in the intermediolateral cell column were not immunoreactive with GABA or glycine antibodies. These results suggest that NADPH diaphorase is largely restricted to GABAergic neurons in the lumbar spinal cord, and that it is mainly present in those neurons in which GABA coexists with glycine or acetylcholine. Since nitric oxide has been implicated in pain processing and hyperalgesia, while GABA, glycine, and acetylcholine are thought to be involved in analgesia and prevention of hyperalgesia, it is likely that nitric oxide synthase-containing GABAergic neurons in dorsal horn have dual actions in transmission of nociceptive information. © 1993 Wiley-Liss, Inc.     

NADPH diaphorase activity and nitric oxide synthase immunoreactivity in lordosis-relevant neurons of the ventromedial hypothalamus

The distribution of the enzymes NADPH diaphorase and nitric oxide synthase in the ventromedial nucleus of the hypothalamus of cycling and ovariectomized/estrogen-treated and control female rats was demonstrated using histochemical and immunocytochemical methods. Serial section analysis of vibratome sections through the entire ventromedial nucleus showed that NADPH diaphorase cellular staining was localized primarily in the ventrolateral subdivision. NADPH diaphorase staining was visible in both neuronal perikarya and processes. Light microscopic immunocytochemistry using affinity-purified polyclonal antibodies to brain nitric oxide synthase revealed a similar pattern of labelling within the ventromedial nucleus and within neurons of the ventrolateral subdivision of the ventromedial nucleus. Control experiments involved omitting the primary antibodies; no labelling was visible under these conditions. Some, but not all, neurons in the ventrolateral subdivision of the ventromedial nucleus contained both NADPH diaphorase and brain nitric oxide synthase as demonstrated by co-localization of these two enzymes in individual cells of this area. That NADPH diaphorase and brain nitric oxide synthase were found in estrogen-binding cells was shown by co-localization of NADPH diaphorase and estrogen receptor and brain nitric oxide synthase and estrogen receptor at the light and ultrastructural levels, respectively. Our studies suggest that brain nitric oxide synthase is present and may be subject to estrogenic influences in lordosis-relevant neurons in the ventrolateral subdivision of the ventromedial nucleus. The hypothalamus is a primary subcortical regulatory center controlling sympathetic function. Therefore, not only is nitric oxide likely to be important for reproductive behavior, but also for the regulation of responses to emotional stress and other autonomic functions.     

Immunoelectron microscopic study of beta-endorphinergic synaptic innervation of GABAergic neurons in the dorsal raphe nucleus.

Using a preembedding double immunoreactive technique by immunostaining with antirat beta-endorphin and antisynthetic glutamic acid decarboxylase antisera sequentially, the synaptic relationships between beta-endorphinergic neuronal fibers and GABAergic neurons in the dorsal raphe nucleus of the rat were examined at the ultrastructural level. Although both beta-endorphin-like immunoreactive fibers and glutamic acid decarboxylase-like immunoreactive neurons can be found in the mediodorsal and medioventral parts of the dorsal raphe nucleus, the synapses between them were found only in the mediodorsal part. Most of the beta-endorphin-like immunoreactive neuronal fibers contained many dense-cored vesicles. The synapses made by beta-endorphin-like immunoreactive neuronal axon terminals on glutamic acid decarboxylase-like immunoreactive neurons were both symmetrical and asymmetrical, with the latter predominant, especially in the axo-dendritic synapses. Perikarya with beta-endorphin-like immunoreactivity were found only in the ventrobasal hypothalamus. These findings suggest the possibility that the beta-endorphin-producing neurons in the ventrobasal hypothalamus could influence GABAergic neurons in the dorsal raphe nucleus directly by synaptic relationships.     

Brainstem afferents to the magnocellular basal forebrain studied by axonal transport, immunohistochemistry, and electrophysiology in the rat

Brainstem afferents to the magnocellular basal forebrain were studied by using tract tracing, immunohistochemistry and extracellular recordings in the rat. WGA-HRP injections into the horizontal limb of the diagonal band (HDB) and the magnocellular preoptic area (MgPA) retrogradely labelled many neurons in the pedunculopontine and laterodorsal tegmental nuclei, dorsal raphe nucleus, and ventral tegmental area. Areas with moderate numbers of retrogradely labelled neurons included the median raphe nucleus, and area lateral to the medial longitudinal fasciculus in the pons, the locus ceruleus, and the medial parabrachial nucleus. A few labelled neurons were seen in the substantia nigra pars compacta, mesencephalic and pontine reticular formation, a midline area in the pontine central gray, lateral parabrachial nucleus, raphe magnus, prepositus hypoglossal nucleus, nucleus of the solitary tract, and ventrolateral medulla. A similar but not identical distribution of labelled neurons was seen following WGA-HRP injections into the nucleus basalis magnocellularis. The possible neurotransmitter content of some of these afferents to the HDB/MgPA was examined by combining retrograde Fluoro-Gold labelling and immunofluorescence. In the mesopontine tegmentum, many retrogradely labelled neurons were immunoreactive for choline acetyltransferase. In the dorsal raphe nucleus, some retrogradely labelled neurons were positive for serotonin and some for tyrosine hydroxylase (TH); however, the majority of retrogradely labelled neurons in this region were not immunoreactive for either marker. The ventral tegmental area, substantia nigra pars compacta, and locus ceruleus contained retrogradely labelled neurons which were also immunoreactive for TH. Of the retrogradely labelled neurons occasionally observed in the nucleus of the solitary tract, prepositus hypoglossal nucleus, and ventrolateral medulla, some were immunoreactive for either TH or phenylethanolamine-N-methyltransferase. To characterize functionally some of these brainstem afferents, extracellular recordings were made from antidromically identified cortically projecting neurons, mostly located in the HDB and MgPA. In agreement with most previous studies, about half (48%) of these neurons were spontaneously active. Electrical stimulation in the vicinity of the pedunculopontine tegmental and dorsal raphe nuclei elicited either excitatory or inhibitory responses in 21% (13/62) of the cortically projecting neurons.(ABSTRACT TRUNCATED AT 400 WORDS)     

Electron microscopic study of GABAergic synaptic innervation of nitric oxide synthase immunoreactive neurons in the dorsal raphe nucleus in the rat

A double immunocytochemical method combining the preembedding avidin biotin peroxidase complex technique and the postembedding immunogold technique was used to examine synaptic interactions between GABAergic and nitric oxide synthase containing neurons in the same tissue sections of the dorsal raphe nucleus of the Wistar white rat. Although a large number of immunogold stained GABAergic axon terminals were found to be presynaptic to dendrites containing nitric oxide synthase-like immunoreaction product, synapses between GABA-like immunoreactive axon terminals and nitric oxide synthase-like immunoreactive perikarya were rare. The labeled boutons were found to make symmetrical and asymmetrical synapses. No axo-axonic synapse was found. These results suggest that GABAergic neurons could modulate nitric oxide producing neurons in the dorsal raphe nucleus through direct synaptic relations. Synapse 25:24–29, 1997. © 1997 Wiley-Liss, Inc.     

Serotoninergic medullary raphespinal projection to the lumbar spinal cord in the rat: a retrograde immunohistochemical study.

Classically the raphespinal system has been regarded as a serotoninergic system; inhibition of spinal nociceptive transmission produced by stimulation of the medullary raphe nuclei is mediated partially by spinal serotoninergic receptors. However, recent evidence suggests that the raphe nuclei are not homogeneous populations of serotoninergic cells. The objective of the present study was to re-examine, in the rat, the serotoninergic raphespinal projection to the lumbar spinal cord, and to determine the relative contribution of serotoninergic raphespinal neurons to the total population of raphespinal neurons. Microinjections of wheat-germ agglutinin horseradish peroxidase conjugate coupled to colloidal gold into the lumbar spinal cord resulted in the retrograde labeling of 53% and 59% of the serotoninergic neurons in the raphe nuclei and in the para-raphe zone, respectively. Conversely, 47% and 28% of the retrogradely labeled neurons in the raphe and para-raphe zone, respectively, demonstrated serotonin-like immunoreactivity. Thus, contrary to previous reports, the present results suggest 1) that only about half of the serotoninergic neurons in the raphe nuclei and in the surrounding para-raphe zone project to the lumbar spinal cord, and 2) that a large proportion of the neurons in the raphe nuclei (53%) and in the surrounding para-raphe zone (72%) that project to the lumbar spinal cord are not serotoninergic.     

Development of catecholaminergic projections to the spinal cord in the North American opossum, Didelphis virginiana

The intent of our study was to determine when catecholaminergic axons grow into each of their adult targets in the spinal cord of the North American opossum (Didelphis virginiana) and to identify the origin of catecholaminergic axons in the lumbosacral cord at different stages of development. Tyrosine hydroxylase-like immunoreactive axons, presumed to be catecholaminergic, were demonstrated at different stages of development by the indirect antibody peroxidase-antiperoxidase technique of Sternberger. The neurons giving rise to such axons in the lumbosacral cord were identified by using the retrograde transport of Fast Blue and immunofluorescence for tyrosine hydroxylase-like immunoreactive neurons. At birth, 12-13 days after conception, tyrosine hydroxylase-like immunoreactive axons are present in the marginal zone throughout the length of the spinal cord. Such axons are particularly numerous in the dorsolateral marginal zone, the region containing most of them in adult animals. By postnatal day 3, a few immunoreactive axons are present in the intermediate (mantle) zone of the spinal cord; and by postnatal day 8, they are most concentrated in the presumptive intermediolateral cell column. Laminae I and II of the dorsal horn are not innervated by such axons until approximately postnatal day 15. By postnatal day 44, the distribution of tyrosine hydroxylase-like immunoreactive axons in the spinal cord resembles that in adult animals, although some areas may be hyperinnervated. At birth, tyrosine hydroxylase-like immunoreactive cell bodies are present in all of the brainstem areas providing catecholaminergic projections to the spinal cord in adult animals (Pindzola et al.: Brain Behav. Evol. 32:281-292, '88); and by at least postnatal day 5, lumbosacral injections of Fast Blue retrogradely label tyrosine hydroxylase-like immunoreactive neurons in all such areas. Retrogradely labeled immunoreactive neurons were also found in areas that do not contain them in adult animals. Such areas include the dorsal part of the nucleus coeruleus and certain areas of the reticular formation. During development, spinally projecting tyrosine hydroxylase-like immunoreactive neurons are numerous medial to the nucleus ventralis lemnisci lateralis (the paralemniscal region), whereas only a few are present in the same location in adult animals. Our results suggest that catecholaminergic axons grow into the spinal cord prenatally, that they innervate their adult targets postnatally and over an extended time period, and that during some stages of development they originate from areas that do not supply them in the adult animal.     

Acute restraint increases NADPH-diaphorase staining in distinct subregions of the rat dorsal raphe nucleus: implications for raphe serotonergic and nitrergic transmission

The brainstem dorsal raphe nucleus (DRN) maintains a rough topographic cell ordering with respect to biological function. This study examined the influence of acute restraint on nitric oxide (NO) synthase (NOS) neurons in distinct DRN subregions. NADPH diaphorase staining (NOS index) intensity was higher in the DRN dorsomedial, ventromedial and lateral wings subregions of restrained vs. control rats. The mean number of cells was not significantly different between both groups of animals. The restrained-induced NADPH-diaphorase activity was significantly higher in the rostral ventromedial and caudal lateral wings than the corresponding caudal and rostral subregions but no significant difference was observed between rostral and caudal dorsomedial subregions. These observations suggest that restraint stress differentially activates NO-producing neurons in distinct DRN subregions.     

Neurotoxic effects of p-chloroamphetamine on the serotoninergic innervation of the trigeminal motor nucleus: a retrograde transport study

The rat forebrain receives projections from both dorsal and median raphe nuclei. It has recently been shown that serotoninergic axons arising from the dorsal raphe nucleus, but not those from the median raphe nucleus, degenerate following systemic administration of p-chloroamphetamine (PCA). The present study was conducted to determine (i) whether the motor nucleus of the trigeminal nerve is innervated by overlapping projections from multiple serotonin cell groups and (ii) whether a particular subset of serotoninergic axon terminals in the trigeminal motor nucleus are sensitive to the neurotoxic effects of PCA. Retrograde transport was used in combination with immunofluorescence to identify the serotonin-positive cells that project to the trigeminal motor nucleus both in control rats and in rats previously treated with PCA. In untreated rats, an average of 95 retrogradely labeled serotonin-positive neurons were found in the dorsal raphe nucleus, 135 in the nucleus raphe obscurus, 132 in the nucleus raphe pallidus and 63 in the ventrolateral medulla. After treatment with PCA, there was a marked decrease (-77%) in the number of retrogradely labeled serotoninergic neurons in the dorsal raphe nucleus, whereas the number of labeled neurons was unchanged in the raphe obscurus and raphe pallidus. These results demonstrate that PCA selectively lesions serotonin axon terminals arising from the dorsal raphe nucleus, while sparing projections from the raphe obscurus and raphe pallidus to the trigeminal motor nucleus. This conclusion is in agreement with previous findings that in the forebrain only axons from the dorsal raphe are vulnerable to PCA. The data provide further evidence that serotoninergic axons originating in the dorsal raphe nucleus differ from other serotoninergic axons in their pharmacological properties and that the dorsal raphe may contain a functionally unique subset of serotonin neurons.     

Brainstem origin of serotonin- and enkephalin-immunoreactive afferents to the opossum's cerebellum

Previous studies have described the distribution of serotonin- and enkephalin-immunoreactive elements in the posterior lobe vermis of the opossum's cerebellum. In the present study we have used a double labeling paradigm which combines the retrograde transport of horseradish peroxidase (HRP) with serotonin and enkephalin immunohistochemistry to determine the brainstem origin of serotoninergic and enkephalinergic neurons that project to the opossum's cerebellar cortex. Subsequent to HRP injections into the posterior lobe vermis, widespread areas of the medulla and pons were found to contain retrogradely labeled neurons. Serotonin-immunoreactive somata are present primarily in the raphe nuclei and the adjacent reticular formation. Enkephalinergic neurons were numerous in the raphe nuclei, medial accessory olive, gigantocellular reticular formation, locus coeruleus, and the nucleus of the trapezoid body. However, serotoninergic neurons that project to the cerebellum were located only in the medullary pyramids and the reticular formation adjacent to the raphe. Double-labeled enkephalinergic neurons were located 1) within the medullary pyramids, 2) throughout the extent of the caudal medial accessory olive, 3) in the rostral subnucleus a of the medial accessory olive, 4) in the nucleus reticularis gigantocellularis pars ventralis, 5) in the nucleus reticularis lateralis, and 6) in the nucleus reticularis ventralis lateral to the inferior olivary complex. These results indicate that although neurons containing serotonin and enkephalin immunoreactivity may be present in some of the same pontine and medullary nuclei, those serotoninergic and enkephalinergic neurons that project to the cerebellum are present primarily in restricted and spatially separate regions of the caudal medulla.     

Expression and colocalization of NADPH-diaphorase and Fos in the subnuclei of the parabrachial nucleus in rats following visceral noxious stimulation

To investigate whether neural nitric oxide synthase (nNOS) in the parabrachial nucleus (PB) is involved in processing visceral noxious stimulation, we mapped the distribution of histochemical staining for nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-d), a marker for nNOS, and immunohistochemical staining for Fos, a neuronal activity marker, in the subnuclei of the PB following 2% formalin injection into the stomach of rats. NADPH-d and noxious-stimuli induced Fos staining were also examined in tissue containing PB cells labeled by the retrograde transport of fluogold (FG) injected into the central nucleus of the amygdala (CeA). We found that the number of Fos immunoreactive (Fos-IR) neurons was significantly increased in the dorsal lateral (dl), external lateral (el) and K?lliker-Fuse (KF) subnuclei of the PB. We observed that intensely labeled (type 1) NADPH-d positive neurons were mainly located in the rostral part of the PB; they extended long processes adjacent Fos-IR neurons, but no Fos/type 1 NADPH-d double-labeled neurons were seen. In contrast, lightly labeled (type 2) NADPH-d positive neurons were principally localized in the dl of the PB, in which a few Fos/type 2 NADPH-d double-labeled neurons were detected. Additionally, a large number of FG/Fos double-labeled neurons were observed to be surrounded closely by the intensive NADPH-d staining in the el of the PB. These results suggest that neurons in the el of the PB that project to the CeA are activated by visceral noxious stimulation and could be indirectly influenced by nitric oxide in the PB.     

Morphology and distribution of serotoninergic and oculomotor internuclear neurons in the cat midbrain

Serotoninergic fibers have been reported in both the abducens and facial nuclei of the cat. Furthermore, serotoninergic dorsal raphe and oculomotor internuclear neurons occupy similar locations in the periaqueductal gray overlying the oculomotor and trochlear motor nuclei. To resolve the issue of whether these two populations of neurons overlap, serotoninergic fibers were assayed in the abducens and facial nucleus; then the morphologies and distributions of identified serotoninergic neurons and oculomotor internuclear neurons were determined. Both the abducens and facial nuclei contained varicosities labelled with antibody to serotonin, but a much higher density of immunoreactive fibers was present in the latter, especially in its medial aspect. Distinct synaptic profiles labelled with antibodies to serotonin were observed in both nuclei. In both cases, terminal profiles contained numerous small, predominantly spheroidal, synaptic vesicles as well as a few, large, dense-core vesicles. These profiles made synaptic contacts onto dendritic and, in the facial nucleus, somatic profiles that occasionally displayed asymmetric, postsynaptic, membrane densifications. Following injection of horseradish peroxidase into either the abducens or facial nuclei, double-label immunohistochemical techniques demonstrated that the serotoninergic and oculomotor internuclear neurons form two distinct cell populations. The immunoreactive serotoninergic cells were distributed within the dorsal raphe nucleus, predominantly caudal to the retrogradely labelled oculomotor internuclear neurons. The latter were located in the oculomotor nucleus along its dorsal border and in the adjacent supraoculomotor area. Intracellular injection of horseradish peroxidase revealed that oculomotor internuclear neurons have multipolar somata with up to ten long, tapering dendrites that bifurcate approximately five times. Their dendritic fields were generally contained within the nucleus and adjacent supraoculomotor area. In contrast, putative serotoninergic neurons were often spindle-shaped and exhibited far fewer primary dendrites. Many of these long, narrow, sparsely branched dendrites crossed the midline and extended to the surface of the cerebral aqueduct. In the vicinity of the aqueduct they branched repeatedly to form a dendritic thicket. The axons of the intracellularly stained serotoninergic neurons emerged either from the somata or the end of a process with dendritic morphology, and in some cases they produced axon collaterals within the periaqueductal gray. Thus the oculomotor internuclear and serotoninergic populations differ in both distribution and morphology.(ABSTRACT TRUNCATED AT 400 WORDS)     

Co-localisation of NADPH diaphorase activity and GABA immunoreactivity in local circuit neurones in the medial prefrontal cortex (mPFC) of the rat

This study provides evidence that neurones in the medial prefrontal cortex of the rat (mPFC areas 24b, 25, and 32) containing strong NADPH diaphorase reactivity also contain GABA immunoreactivity. Also demonstrated is the co-localisation of NADPH diaphorase activity with immunoreactivity for the neuronal isoform of nitric oxide synthase (nNOS) in mPFC neurones. Qualitative and quantitative analyses in the light and electron microscopes indicate that strongly NADPH diaphorase reactive cells are a subpopulation of GABAergic local circuit neurones and constitute a very small proportion (0.6–1.1%) of neurones in rat mPFC. These results suggest that NADPH diaphorase reactive cells in rat mPFC can influence neural activity via GABA-mediated and NO-mediated mechanisms.