Enkephalinergic innervation of GABAergic neurons in the dorsal raphe nucleus of the rat

The preembedding double immunoreaction method was used to study interrelations of enkephalinergic and GABAergic neuronal elements in the dorsal raphe nucleus of the Wistar albino rat. The enkephalin-like neuronal elements were immunoreacted by the peroxidase-antiperoxidase method and silver-gold intensified, which showed strongly and was specific. The GABA-like immunoreactive neurons were immunoreacted by the peroxidase-antiperoxidase method only. GABA-like neural somata were postsynaptic to both the enkephalin-like immunoreactive and the non-immunoreactive axon terminals. The enkephalin-like immunoreactive axon terminals were also found to synapse GABA-like immunoreactive dendrites. The GABA-like immunoreactive neuronal elements were also found to receive synapses from other non-immunoreactive as well as GABA-like immunoreactive axon terminals. Almost all of the synapses appeared to be asymmetrical. Possible functional activity of interactions among the enkephalinergic, GABAergic, and serotonergic neuronal elements in the dorsal raphe nucleus are discussed.     

Synaptic relations of neurotensinergic neurons in the dorsal raphe nucleus

The ultrastructure and synaptic relations of neurotensinergic neurons in the rat dorsal raphe nucleus (DRN) were examined. The neurotensin-like immunoreactive (NT-L1) neurons in the DRN were fusiform or spherical. The NT-LI perikarya could only be detected in colchicine-treated animals whereas the immunoreactive axon terminals could only be found in the animals not treated with colchicine. Although many NT-LI dendrites received synapses from nonimmunoreactive axon terminals, the NT-LI perikarya received few synapses. NT-LI axon terminals also made synapses on nonimmunoreactive dendrites. Occasionally, synapses were found between the NT-LI axon terminals and NT-LI dendrites in the cases in which the animals were not treated with colchicine.     

Nitric oxide synthase immunoreactivity in rat pontine medullary neurons

Nitric oxide synthase immunoreactivity was detected in neurons and fibers of the rat pontine medulla. In the medulla, nitric oxide synthase-positive neurons and processes were observed in the gracile nucleus, spinal trigeminal nucleus, nucleus of the solitary tract, dorsal motor nucleus of the vagus, nucleus ambiguus, medial longitudinal fasciculus, reticular nuclei and lateral to the pyramidal tract. In the pons, intensely labeled neurons were observed in the pedunculopontine tegmental nucleus, paralemniscal nucleus, ventral tegmental nucleus, laterodorsal tegmental nucleus, and lateral and medial parabrachial nuclei. Labeled neurons and fibers were seen in the interpeduncular nuclei, dorsal and median raphe nuclei, central gray and dorsal central gray, and superior and inferior colliculi. Double-labeling techniques showed that a small population (< 5%) of nitric oxide synthase-positive neurons in the medulla also contained immunoreactivity to the aminergic neuron marker tyrosine hydroxylase. The majority of nitric oxide synthase-immunoreactive neurons in the dorsal and median raphe nuclei were 5-hydroxytryptamine-positive, whereas very few 5-hydroxytryptamine-positive cells in the caudal raphe nuclei were nitric oxide synthase-positive. Virtually all nitric oxide synthase-positive neurons in the pedunculopontine and laterodorsal tegmental nuclei were also choline acetyltransferase-positive, whereas nitric oxide synthase immunoreactivity was either low or not detected in choline acetyltransferase-positive neurons in the medulla. The results indicate a rostrocaudal gradient in the intensity of nitric oxide synthase immunoreactivity, i.e. it is highest in neurons of the tegmentum nuclei and neurons in the medulla are less intensely labeled. The majority of cholinergic and serotonergic neurons in the pons are nitric oxide synthase-positive, whereas the immunoreactivity was either too low to be detected or absent in the large majority of serotonergic, aminergic and cholinergic neurons in the medulla.     

Direct evidence for nitric oxide synthase in vagal afferents to the nucleus tractus solitarii

The anatomical relationship between vagal afferents and brain nitric oxide synthase containing terminals in the nucleus tractus solitarii was studied by means of anterograde tracing combined with immunocytochemistry and immuno-electron microscopy. Biotinylated dextran amine was injected into the nodose ganglion with a glass micropipette. Four to eight days following the injection, regions of the nucleus tractus solitarii containing biotinylated dextran amine-labelled vagal afferents and those containing nitric oxide synthase-immunopositive terminals were congruent. Many neurons exhibiting nitric oxide synthase immunoreactivity were found within the biotinylated dextran amine-containing terminal field. However dense labeling of terminals with biotinylated dextran amine precluded determination if the terminals were nitric oxide synthase-immunoreactive. Therefore, we combined degeneration of vagal afferents after removal of one nodose ganglion with nitric oxide synthase immuno-electron microscopy. Axon terminals that possessed characteristic vesicle clusters and were partially or completely engulfed by glial processes were identified as degenerating vagal afferents. Degenerating axon terminals comprised 38% of the total axon terminals in the nucleus tractus solitarii in a sample of sections; and of the degenerating axon terminals, 67% were nitric oxide synthase-immunoreactive. Nitric oxide synthase immunoreactivity was present in 41% of the non-degenerating axon terminals. Prominent staining of dendrites for nitric oxide synthase immunoreactivity indicated that much of the nitric oxide synthase in the nucleus tractus solitarii is not derived from peripheral afferents. Of the total number of dendritic profiles sampled, half were nitric oxide synthase-immunoreactive. Our data support the hypothesis that nitric oxide or nitric oxide donors may be present in primary vagal afferents that terminate in the nucleus tractus solitarii. While this study confirms that vagal afferents contain brain nitric oxide synthase, it demonstrates for the first time that the majority of nitric oxide synthase immunoreactivity in the nucleus tractus solitarii is found in intrinsic structures in the nucleus. In addition, our data show that second or higher order neurons in the nucleus tractus solitarii may be nitroxidergic and receive both nitroxidergic and non-nitroxidergic vagal input.     

Beta-endorphinergic innervation of mu and delta receptor containing neurons in the dorsal raphe nucleus

A pre-embedding double immunostaining technique was used to determine the role of beta-endorphin in synapse, particularly in neurons with a postsynaptic membrane containing micro-1 or delta-1 opioid receptors. A small number of beta-endorphin immunoreactive axon terminals in the dorsal raphe nucleus was found to make direct synapses on micro-1 or delta-1 opioid receptor-immunoreactive dendrites, some of which showed immunostaining of their postsynaptic membranes, although with low frequencies. These results suggest that beta-endorphin can play a direct role through the micro-1 or delta-1 opioid receptors at synapses, but the main route would be through other opioid receptor at the synapse or even not through the synapse.     

Release of cerebral 5-hydroxytryptamine evoked by electrical stimulation of the dorsal and median raphe nuclei: effect of a neurotoxic amphetamine

Recent neuroanatomical data suggest that the axons and terminals of serotonergic neurons of the dorsal and median raphe nuclei are morphologically and pharmacologically distinct. Here we attempted to establish a functional in vivo model of serotonergic terminals derived from these nuclei, and then carry out a preliminary comparison of their physiological and pharmacological properties. Brain microdialysis was used to monitor extracellular 5-hydroxytryptamine in the hippocampus (dorsal and median raphe innervation) and frontal cortex (preferential dorsal raphe innervation) of the anaesthetized rat. To distinguish 5-hydroxytryptamine released by terminals of dorsal raphe neurons from that released by median raphe neurons, one or other of these nuclei was stimulated electrically. Electrical stimulation of either the dorsal or median raphe nucleus evoked a release of 5-hydroxytryptamine in the hippocampus. Whereas stimulation of the dorsal raphe nucleus also released 5-hydroxytryptamine in the frontal cortex, stimulation of the median raphe nucleus did not. No release of 5-hydroxytryptamine was evoked when electrodes were located in regions bordering the dorsal raphe nucleus and the median raphe nucleus. The amounts of hippocampal 5-HT released by stimulation of the dorsal or median raphe nucleus were found to be similarly altered by a 5-hydroxytryptamine uptake inhibitor (citalopram) and calcium-free perfusion medium, and also by increasing stimulation frequency (2-10 Hz). Furthermore, the amount of 5-hydroxytryptamine released by electrical stimulation of either the dorsal raphe nucleus or median raphe nucleus was markedly reduced in rats pretreated with p-chloroamphetamine. In summary, our data show that electrical stimulation of the dorsal or median raphe nucleus releases 5-hydroxytryptamine in a regionally specific manner (hippocampus versus frontal cortex), suggesting that serotonergic nerve terminals of the dorsal and median raphe pathways were being activated selectively. Using this model, we found no differences in the responsiveness of dorsal and median raphe pathways to a specific set of physiological and pharmacological manipulations. In particular, our data suggest that the neurotoxic action of p-chloroamphetamine may not be targeted solely on serotonergic axons and terminals of the dorsal raphe nucleus but includes those of the median raphe nucleus.     

Does licensing of drugs in industrialized countries guarantee drug quality and safety for Third World countries? The case of norplant licensing in Finland

A knowledge of the anatomy of medullary serotonergic cells is critical to understanding local and brainstem circuits in which these cells participate. Serotonergic neurons (n = 16) were identified, as previously described (Mason [1997] J. Neurophysiol. 77:1087-1098) by their slow and steady background discharge in halothane anesthetized rats. Neurons were then intracellularly labeled with Neurobiotin and visualized with 3,3'diaminobenzidine. The validity of the physiological identification of serotonergic cells was confirmed by processing two neurons that were physiologically characterized as serotonergic for serotonin immunoreactivity; both tested cells contained immunoreactive serotonin. The dendrites and axon of each labeled cell were reconstructed by using a three-dimensional computerized system. Somata were small or medium in size and had fusiform, triangular, or multipolar shapes. The dendritic arbor was constricted with most dendrites extending for less than 500 microm from the soma. All labeled axons projected caudally and travelled in the ventrolateral medulla, either dorsal or ventral to the lateral reticular nucleus. Most cells had collaterals and/or dense axonal swellings in the nucleus reticularis gigantocellularis, nucleus reticularis magnocellularis, raphe magnus, and the ventrolateral medulla. Non-local collaterals and swellings were also observed in the nucleus reticularis gigantocellularis and in the ventrolateral medulla at all medullary levels. The results demonstrate that 1) the dendrites of serotonergic cells are restricted to raphe magnus and the ventral part of nucleus reticularis magnocellularis; and 2) serotonergic cells project to medullary nuclei that contain bulbospinal cells which project to dorsal, intermediate, and ventral horns. Serotonergic cell projections to brainstem sites may mediate the integration of sensory, autonomic, and motor modulation at the brainstem level.     

Electron microscopic data on the neurons of nuclei subpretectalis and posterior-ventralis thalami. A combined immunohistochemical study

The nucleus rotundus receives GABA-like immunoreactive fibres from the nuclei subpretectalis and postero-ventralis thalami. This result was confirmed by Phaseolus vulgaris leucoagglutinin (PhA-L) anterograde tracer and with electron microscopic (EM) gamma-aminobutiric acid (GABA)-immunogold staining. The detailed electron microscopic analysis of the structure of the neurons in these nuclei revealed that the neurons in the nucleus subpretectalis displayed GABA-like immunoreactivity. In the postero-ventral thalamic nucleus a group of neurons was GABA-positive. The surface of the neurons was covered both with numerous GABA-negative and GABA-like immunoreactive terminals that established asymmetrical and symmetrical synapses, respectively, with the GABA-positive neurons. The GABA-like immunonegative terminals are supposed to be the axon terminals of the collaterals of tecto-rotundal fibres in the subpretectal nucleus and the collateral terminal branches of contralateral tecto-rotundal fibres in the postero-ventralis thalami. In both nuclei, the GABA-like immunoreactive terminals may be developed by the collaterals of local neurons that establish symmetrical synapses. In the Phaseolus lectin-stained preparations these terminals may be labelled. The morphological characteristics of the neurons in the subpretectal and partly, in the posteroventral nuclei are similar to those of interneurons (local circuit neurons) and the numerous asymmetrical and symmetrical axo-somatic synapses, respectively. But these neurons locate outside of their target nucleus, and exert their modulatory effect on rotundo-ectostriatal transmission. Also, a contralateral influence is present in the nucleus rotundus that may interact in the cooperation of the eyes. The neurons of the subpretectal and posteroventral nuclei, similarly to the neurons of isthmic nuclei, are a special group of modulatory neurons with effects at a distance.     

A haplotype and linkage disequilibrium analysis of the hereditary hemochromatosis gene region

Monoclonal antibodies were generated against serotonin (5-HT) and the C-terminal portion of the neuronal form of nitric oxide synthase (nNOS), the enzyme producing nitric oxide in neurons. These antibodies were used to compare the distribution of 5-HT- and nNOS-containing neurons in the raphe nuclei of four animal species (rat, mouse, guinea pig, and cat). It was found that the rat was the only species in which the raphe nuclei contain a substantial number of nNOS-immunoreactive (IR) cell bodies. In this species and as observed by other authors, all mesencephalic raphe nuclei contained nNOS-IR cells, the largest group being located in the nucleus raphe dorsalis. The coexistence of nNOS and 5-HT immunoreactivities in these nuclei was visualized by double labeling. In the medulla, the nuclei raphe magnus and obscurus displayed a rather low number of nNOS-IR neurons. In the other species, nNOS-IR cell bodies were found in very low numbers, whatever raphe nucleus was considered. The rostral pole of the nucleus raphe dorsalis and the nuclei raphe magnus and obscurus contained a few nNOS-IR neurons which did not show any coincidence with the 5-HT neurons. In addition, nNOS-IR axons were rare. It is concluded that in the mouse, guinea pig, and cat the involvement of nitric oxide in functions subserved by 5-HT within the raphe nuclei might be minimal.     

Origin of projections from the midbrain raphe nuclei to the hypothalamic paraventricular nucleus in the rat: a combined retrograde and anterograde tracing study

A number of neuronal functions governed by the hypothalamic paraventricular nucleus are influenced by serotonin, and it is generally believed that the moderate density of serotonin-immunoreactive fibres and terminals within the paraventricular nucleus originates from the midbrain dorsal and median raphe nuclei. To further evaluate the intricate anatomy of projections from brain stem raphe nuclei of the rat, a combination of retrograde and anterograde tracing experiments were conducted to determine the medullary raphe nuclei projection to the paraventricular nucleus. Rhodamine-labelled latex microspheres, Cholera toxin subunit B and FluoroGold we used as retrograde tracers. Intracerebroventricular injections into the third ventricle of all retrograde tracers labelled a distinct population of neurons in the dorsal raphe situated in the subependymal stratum adjacent to the cerebral aqueduct indicating that these cells take up the tracer from the cerebrospinal fluid. Very few retrogradely labelled neurons were seen in the median raphe after i.c.v. administration of the tracers. Retrograde tracers delivered into the medial part of the paraventricular nucleus labelled no further cells in the midbrain dorsal and median raphe nuclei, whereas a substantial number of retrogradely labelled cells emerged in the pontine raphe magnus. However, when the retrograde tracers were delivered into the lateral part of the paraventricular nucleus, avoiding leakage of the tracer into the ventricle, very few labelled neurons were seen in the dorsal and median raphe, whereas the prominent labelling of raphe magnus neurons persisted. The anatomical organization of nerve fibres terminating in the area of the paraventricular nucleus originating from midbrain raphe nuclei was studied in a series of anterograde tracing experiments using the plant lectin Phaseolus vulgaris leucoagglutinin. Injections delivered into the dorsal raphe or median raphe labelled but a few fibres in the paraventricular nucleus proper. A high number of fine calibered nerve fibres overlying the ependyma adjacent to the paraventricular nucleus was, however, seen after the injections into the subependymal rostral part of the dorsal raphe. Injections delivered into the raphe magnus gave rise to a dense plexus of terminating fibres in the parvicellular parts of the paraventricular nucleus and moderately innervated the posterior magnocellular part of the paraventricular nucleus as well as the magnocellular supraoptic nucleus. Concomitant visualization of serotonin-immunoreactive neurons and retrograde FluoroGold-tracing from the paraventricular nucleus revealed that none of the serotonergic neurons of the raphe magnus projects to this nucleus, while a few of the neurons putatively projecting to the paraventricular nucleus from the median raphe are serotonergic. The current observations suggest that the raphe magnus constitute by far the largest raphe input to the paraventricular nucleus and strongly questions the earlier held view that most raphe fibres innervating the paraventricular nucleus are derived from the midbrain dorsal and median raphe. However, the source of serotonergic innervation of the paraventricular nucleus remains elusive.     

Immunocytochemical and histochemical localization of nitric oxide synthase in the turtle retina

Recent interest in nitric oxide and its relationship to cGMP has produced many attempts to anatomically localize the enzyme synthesizing nitric oxide, nitric oxide synthase. In the retina, numerous previous studies have used the NADPH-diaphorase enzyme activity of nitric oxide synthase as a histochemical method to localize nitric oxide synthase. However, all NADPH-diaphorase activity is not necessarily nitric oxide synthase, because several enzymes have similar biochemical activity. Additionally, various histochemical methods have been used to demonstrate NADPH-diaphorase activity, which makes comparisons between studies difficult. The purpose of this study was twofold. First, we wanted to examine the histochemical labeling of NADPH-diaphorase in the turtle retina to allow comparisons to previous studies. Second, we wanted to compare the histochemical localization of NADPH-diaphorase activity to the immunocytochemical localization of nitric oxide synthase in the turtle retina. Our histochemical localization of NADPH-diaphorase activity and our localization of nitric oxide synthase-like immunoreactivity in the turtle retina both produced similar results. Both the histochemistry and immunocytochemistry consistently labeled photoreceptor inner segments, at least three amacrine cell types, and processes in the inner plexiform layer. In optimized double-labeled preparations, all cells with NADPH-diaphorase activity were also positive for nitric oxide synthase-like immunoreactivity, although some somata in the ganglion cell layer only had nitric oxide synthase-like immunoreactivity. The immunocytochemical localization of nitric oxide synthase in photoreceptors, amacrine cells, and putative ganglion cells indicates that nitric oxide may function at several levels of visual processing in the turtle retina.     

Ultrastructural localization of nitric oxide synthase immunoreactivity in the cat ventrobasal complex

This study describes the ultrastructural localization of nitric oxide synthase (NOS) immunoreactivity in the cat ventrobasal complex. NOS immunoreactivity was found in the cell bodies and dendrites of local circuit neurons and in vesicle-containing profiles. The vesicle-containing profiles could be divided into two classes, those of dendritic origin (presynaptic dendrite boutons) and those of axonal origin. The NOS labelled axon terminals varied in size and packing density and were principally located in the extra-glomerular neuropil. These boutons presented a range of morphologies and it was not possible to determine the probable source based on morphological criteria. The NOS immunoreactive presynaptic dendrite boutons were found both within and outside glomeruli and established both pre- and post-synaptic relationships with other elements. Post-embedding GABA immunocytochemistry showed that some NOS immunoreactive axonal boutons and presynaptic dendrites were also immunopositive for GABA. This finding suggests that some of the NOS labelled axonal boutons are of local circuit neuron origin. These results suggest that local circuit neurons in the cat ventrobasal complex might be involved in specific, short range interactions using GABA and longer, more global interactions using nitric oxide.     

Topographical distribution of NADPH-diaphorase activity in the central nervous system of the frog, Rana perezi

The distribution of NADPH-diaphorase (ND) activity was histochemically investigated in the brain of the frog Rana perezi. This technique provides a highly selective labeling of neurons and tracts. In the telencephalon, labeled cells are present in the olfactory bulb, pallial regions, septal area, nucleus of the diagonal band, striatum, and amygdala. Positive neurons surround the preoptic and infundibular recesses of the third ventricle. The magnocellular and suprachiasmatic hypothalamic nuclei contain stained cells. Numerous neurons are present in the anterior, lateral anterior, central, and lateral posteroventral thalamic nuclei. Positive terminal fields are organized in the same thalamic areas but most conspicuously in the visual recipient plexus of Bellonci, corpus geniculatum of the thalamus, and the superficial ventral thalamic nucleus. Labeled fibers and cell groups are observed in the pretectal area, the mesencephalic optic tectum, and the torus semicircularis. The nuclei of the mesencephalic tegmentum contain abundant labeled cells and a conspicuous cell population is localized medial and caudal to the isthmic nucleus. Numerous cells in the rhombencephalon are distributed in the octaval area, raphe nucleus, reticular nuclei, sensory trigeminal nuclei, nucleus of the solitary tract, and, at the obex levels, the dorsal column nucleus. Positive fibers are abundant in the superior olivary nucleus, the descending trigeminal, and the solitary tracts. In the spinal cord, a large population of intensely labeled neurons is present in all fields of the gray matter throughout its rostrocaudal extent. Several sensory pathways were heavily stained including part of the olfactory, visual, auditory, and somatosensory pathways. The distribution of ND-positive cells did not correspond to any single known neurotransmitter or neuroactive molecule system. In particular, abundant codistribution of ND and catecholamines is found in the anuran brain. Double labeling techniques have revealed restricted colocalization in the same neurons and only in the posterior tubercle and locus coeruleus. If ND is in amphibians a selective marker for neurons containing nitric oxide synthase, as generally proposed, with this method the neurons that may synthesize nitric oxide would be identified. This study provides evidence that nitric oxide may be involved in novel tasks, primarily related to forebrain functions, that are already present in amphibians.     

Role of nitric oxide in medullary raphe-evoked inhibition of neuronal activity in the periaqueductal gray matter

In male urethane-anaesthetized rats, activation of neurons in nucleus raphe obscurus and the caudal tip of nucleus raphe magnus by microinjection of 50-100 nl 0.1 M D,L-homocysteic acid produced a 75.6 +/- 5.2% reduction in the firing rate in 25 neurons in the lateral and dorsolateral sectors of the periaqueductal gray matter which lasted for 102.3 +/- 13.3s (mean +/- S.E.M.). The duration of the inhibition was significantly reduced in a dose-dependent manner by intracerebroventricular injection of the inhibitor of nitric oxide synthase, N(w)-nitro-L-arginine methyl ester (50-500 micrograms) but not by N(w)-nitro-D-arginine methyl ester (500 micrograms). In contrast, the magnitude of the raphe-evoked inhibition, i.e. the maximum depression of firing rate, was not significantly affected by either isomer. The results suggest that nitric oxide plays a role in the regulation of the excitability of neurons in the midbrain aversive system by the medullary raphe. The selective effect of the nitric oxide synthase inhibitor on the duration, but not the magnitude, of the raphe-evoked inhibition suggests that nitric oxide is not involved in initiating the inhibition. Rather, its role appears to be in maintaining the raphe-evoked inhibition once it has been initiated by a non-nitrergic mechanism.     

Oxytocinergic and serotonergic innervation of identified lumbosacral nuclei controlling penile erection in the male rat

Penile erection is due to activation of proerectile neurons located in the sacral parasympathetic nucleus of the L6-S1 spinal cord in the rat. Contraction of the ischiocavernosus and bulbospongiosus striated muscles, controlled by motoneurons located in the ventral horn of the L5-L6 spinal cord, reinforces penile erection. Physiological and pharmacological arguments have been provided for a role of oxytocin and serotonin in the spinal regulation of penile erection. Immunohistochemistry of oxytocinergic and serotonergic fibres was performed at the lumbosacral level of the male rat spinal cord, and combined with retrograde tracing from the pelvic nerve or from the ischiocavernosus and bulbospongiosus muscles using wheat germ agglutinin-horseradish peroxidase. Sacral preganglionic neurons retrogradely labelled from the pelvic nerve formed a homogeneous population, predominant at the L6 level. Motoneurons retrogradely labelled from the ischiocavernosus and bulbospongiosus muscles were observed in the medial part of the dorsolateral and in the dorsomedial nuclei. Fibres immunoreactive for oxytocin were mainly distributed in the superficial layers of the dorsal horn, the dorsal gray commissure and the sacral parasympathetic nucleus. Some of these fibres were apposed to retrogradely-labelled sacral preganglionic neurons and at the ultrastructural level, some synapses were evidenced. Fibres immunoreactive for serotonin were largely and densely distributed in the dorsal horn, the dorsal gray commissure, the sacral parasympathetic nucleus and the ventral horn. Some serotonergic fibres occurred in close apposition with retrogradely-labelled sacral preganglionic neurons and motoneurons, and synapses were demonstrated at the ultrastructural level. This study provides morphological support for a role of oxytocin and serotonin on sacral preganglionic neurons innervating pelvic organs and motoneurons innervating the ischiocavernosus and bulbospongiosus muscles.     

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.     

Distribution of nitric oxide synthase in the esophagus of the cat and monkey

The distribution of nitrergic neurons and processes in the esophagus of the cat and monkey was studied by light microscopic immunocytochemistry using a specific antibody against purified rat brain nitric oxide synthase and immunoperoxidase procedures. Immunoreactive nerve fibers were found pervading the myenteric plexus, submucous plexus and plexus of the muscularis mucosae, and particularly in the lower esophagus a few immunoreactive fibers entered the epithelium as free nerve endings, some of which derived from perivascular fibers. In the upper esophagus immunoreactive motor end-plates were found in the striated muscle. Thirty-forty-five percent of neuronal cell bodies found in the intramural ganglia and along the course of nerve fiber bundles were immunoreactive and were of the three morphological types earlier described. In the intramural ganglia immunoreactive nerve fibers formed a plexus in which varicose nerve terminals were in close relation to immunoreactive and non-immunoreactive neurons. The intramural blood vessels that crossed the different layers of the esophageal wall were surrounded by paravascular and perivascular plexuses containing immunoreactive nerve fibers. The anatomical findings suggest that nitric oxide is involved in neural communication and in the control of peristalsis and vascular tone in the esophagus. In the lower esophagus a few nitrergic nerve fibers are anatomically disposed to subserve a sensory-motor function.     

Characterization of cytokine and iNOS mRNA expression in situ during the course of experimental autoimmune myocarditis in rats

The distribution of GABAergic elements and their synaptic contacts in the nucleus submedius, a specific nociceptive relay in the medial thalamus of the cat, was studied using light and electron-microscopic postembedding immunohistochemical methods. About one-fourth of the neurons in nucleus submedius were GABA immunoreactive. These neurons were generally smaller than the unlabeled neurons and are probably local circuit neurons. Electron microscopy showed GABA immunoreactivity in two types of vesicle-containing profiles, F-terminals and presynaptic dendrites. F-terminals formed simple synapses with the dendrites of presumed thalamocortical relay cells. Presynaptic dendrites were involved in more complex synaptic arrangements that included ascending trigeminothalamic and spinothalamic tract terminals and thalamocortical relay cell dendrites. Analysis of single sections showed that about 40% of the trigeminothalamic and spinothalamic tract terminals, identified by anterograde transport of horseradish peroxidase, were presynaptic to GABAergic presynaptic dendrites. These results show that GABAergic neurons are frequent in nucleus submedius and that the GABAergic elements make synaptic connections similar to those described for other sensory relay nuclei, including the somatosensory ventroposterior nucleus. This suggests that GABAergic mechanisms play an important role in the processing of nociceptive and thermoreceptive information.     

Synaptic innervation of enkephalinergic neurons by axon terminals immunoreactive to dopamine-beta-hydroxylase in the rat area postrema

A preembedding double immunostaining technique was used to study synaptic relations between enkephalin-like immunoreactive and dopamine-beta-hydroxylase-like immunoreactive neurons in the rat area postrema. Enkephalin-like immunoreactive neuronal perikarya and dendrites were found to receive synapses from dopamine-beta-hydroxylase-like immunoreactive axon terminals. Synapses were also found between the same dopamine-beta-hydroxylase-like immunoreactive neurons. Compared with our previous study, the present results provide morphological evidence that dopaminergic and noradrenergic neurons have different synaptic relations with enkephalinergic neurons, suggesting that physiological functions, especially those related to enkephalinergic neurons, may be different from each other in the area postrema.     

Co-localization of serotonin and FMRFamide-like immunoreactivities in the central nervous system of the earthworm, Eisenia fetida

In addition to receptor-type pinealocytes, the mammalian pineal organ contains small and large neurons and ependymal/glial cells as well. Axons of pinealocytes form synaptic ribbon-containing axo-dendritic synapses on large secondary pineal neurons and/or terminate as neurohormonal endings on the basal lamina of the vascular surface of the organ. The small pineal neurons were found to be gamma-aminobutyric acid (GABA)-immunoreactive, while large secondary neurons and pinealocytes contained immunoreactive amino acids (glutamate and aspartate). Glutamate accumulated presynaptically in pinealocytic axon terminals on large secondary neurons and in the axons of these neurons. Glutamate immunoreactive axons of pineal neurons were traced via the pineal tract to the habenular nucleus. Axons containing granular vesicles and coming from extrapineal perikarya are glutamate immunoreactive as well. Aspartate and GABA are also present in some of the myelinated axons, supposedly pinealopetal in the pineal tract.