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.     

Differential effects of 5-HT1B/1D receptor antagonists in dorsal and median raphe innervated brain regions

The present study has employed in vitro electrophysiology to characterise the ability of bradykinin to depolarise the rat isolated nodose ganglion preparation, containing the perikarya of vagal afferent neurons. Both bradykinin and kallidin elicited a concentration-dependent (1-100 nM) depolarisation when applied to the superfusate bathing the nodose ganglia, whereas the bradykinin B1 receptor agonist, des-Arg9-bradykinin, was only effective in the micromolar range. Furthermore, the electrophysiological response to bradykinin was antagonised by the bradykinin B2 receptor antagonist, D-arginyl-L-arginyl-L-prolyl-trans-4-hydroxy-L-prolylglycyl-3-(2-t hienyl)-L-alanyl-L-seryl-D-1,2,3,4-tetrahydro-3-isoquinolinecarbonyl+ ++-L-(2alpha,3beta,7abeta)-octahydro-1H-indole-2-carbonyl-L- arginine (Hoe 140), in a concentration-related manner. To determine the anatomical location of functional bradykinin B2 receptors, in vitro autoradiography with [125I]para-iodophenyl Hoe 140 was performed on sections of rat and human inferior vagal (nodose) ganglia and confirmed the presence of binding over vagal perikarya. Collectively, these data provide evidence for functionally relevant bradykinin B2 receptors on vagal afferent neurons, which are apparently also present on human vagal perikarya.     

Functional GABAA receptors on rat vagal afferent neurones

1. In the present study, in vitro electrophysiology and receptor autoradiography were used to determine whether rat vagal afferent neurones possess gamma-aminobutyric acid (GABA)A receptors. 2. GABA (1-100 microM) and isoguvacine (3-100 microM) caused a concentration-dependent depolarization of the rat isolated nodose ganglion preparation at room temperature. When applied to the tissue 20 min before the agonist, SR95531 (3 microM) and bicuculline (3 microM) caused a parallel shift to the right of the GABA and isoguvacine concentration-response curves, yielding shifts of 81 fold and 117 fold for SR95531 and 4 fold and 12 fold for bicuculline, respectively. 3. Baclofen (10 nM-100 microM) was unable to elicit a depolarization of the rat isolated nodose ganglion preparation at either room temperature or at 36 degrees C, whilst 5-aminovaleric acid (10 microM), a GABAB receptor antagonist, was unable to antagonize significantly the GABA-induced depolarization at either room temperature or at 36 degrees C. 4. [3H]-SR95531 (7.2 nM), a GABAA receptor-selective antagonist, bound topographically to sections of rat brainstem. Specific binding was highest in the medial nucleus tractus solitarius (NTS) and dorsal motor nucleus of the vagus nerve (DMVN). Binding was also observed in certain medullary reticular nuclei, in particular the parvocellular reticular nucleus. 5. Unilateral nodose ganglionectomy caused a reduction in GABAA binding site density in the medial NTS from 93 +/- 7 to 68 +/- 6 d.p.m./mm2. This procedure also caused a reduction in GABAA binding site density in the side of the NTS contralateral to the lesion, from 151 +/- 12 to 93 +/- 7 d.p.m./mm2. Sham surgery had no effect on the binding of [3H]-SR95531 in rat brainstem. 6. The present data provide evidence for the presence of GABAA receptors located on the soma and central terminals of rat vagal afferent neurones. Additionally, a population of GABAA receptors is evidenced postsynaptically in the rat NTS with respect to vagal afferent terminals. These data are discussed in relation to the functional pharmacology of GABA in this region of the NTS.     

Vagal efferent and afferent innervation of the rat esophagus as demonstrated by anterograde DiI and DiA tracing: focus on myenteric ganglia

Anterograde tracing with the carbocyanine tracer DiI and the aminostyrol derivative DiA was used to selectively label fibers from the nucleus ambiguus, dorsal motor nucleus and nodose ganglion, respectively, terminating in the rat esophagus, and to compare them with the innervation of the gastric fundus in the same animals. Ambiguus neurons terminated on motor endplates distributed mainly to the ipsilateral half of the esophagus. There was no evidence of preganglionic innervation of myenteric ganglia from ambiguus neurons. Neurons of the dorsal motor nucleus supplied sparse fibers to only about 10% of enteric ganglia in the esophagus while they innervated up to 100% of myenteric ganglia in the stomach. Neurons of the nodose ganglion terminated profusely on more than 90% of myenteric ganglia of the esophagus and on about 50% of ganglia in the stomach. Afferent vagal fibers were also frequently found in smooth muscle layers starting at the esophago-gastric junction. In contrast, they were extremely rare in the striated muscle part of the esophagus. These morphological data suggest a minor influence of neurons of the dorsal motor nucleus and a prominent influence of vagal afferent terminals onto myenteric neurons in the rat esophagus.     

Adolescent abstinence and condom use: are we sure we are really teaching what is safe?

Previous anatomical studies demonstrated vagal innervation to the ovary and distal colon and suggested the vagus nerve has uterine inputs. Recent behavioral and physiological evidence indicated that the vagus nerves conduct sensory information from the uterus to the brainstem. The present study was undertaken to identify vagal sensory connections to the uterus. Retrograde tracers, Fluorogold and pseudorabies virus were injected into the uterus and cervix. DiI, an anterograde tracer, was injected into the nodose ganglia. Neurectomies involving the pelvic, hypogastric, ovarian and abdominal vagus nerves were performed, and then uterine whole-mounts examined for sensory nerves containing calcitonin gene-related peptide. Nodose ganglia and caudal brainstem sections were examined for the presence of estrogen receptor-containing neurons in "vagal locales." Labeling of uterine-related neurons in the nodose ganglia (Fluorogold and pseudorabies virus) and in the brainstem nuclei (pseudorabies virus) was obtained. DiI-labeled nerve fibers occurred near uterine horn and uterine cervical blood vessels, in the myometrium, and in paracervical ganglia. Rats with vagal, pelvic, hypogastric and ovarian neurectomies exhibited a marked decrease in calcitonin gene-related peptide-immunoreactive nerves in the uterus relative to rats with pelvic, hypogastric, and ovarian neurectomies with intact vagus nerves. Neurons in the nodose ganglia and nucleus tractus solitarius were immunoreactive for estrogen receptors. These results demonstrated: (1) the vagus nerves serve as connections between the uterus and CNS, (2) the nodose ganglia contain uterine-related vagal afferent neuron cell bodies, and (3) neurons in vagal locales contain estrogen receptors.     

Glutamate, gamma-aminobutyric acid and tachykinin-immunoreactive synapses in the cat nucleus tractus solitarii

Neurophysiological and pharmacological evidence suggests that glutamate, gamma-aminobutyric acid and tachykinins (substance P and neurokinin A) each have a role in cardiovascular regulation in the nucleus tractus solitarii. This study describes the ultrastructural relationships between nerve terminals immunoreactive for these substances in the nucleus tractus solitarii of the cat using post-embedding immunogold (single and double) labelling techniques on sections of tissue embedded in LR White resin. The technique combines a high specificity of labelling with good ultrastructural and antigenic preservation. Glutamate-immunoreactive terminals, recognized by their high density of gold particle labelling compared to the mean tissue level of labelling, accounted for about 40% of all synaptic terminals in the region of the nucleus tractus solitarii analysed (medial, dorsal, interstitial, gelatinosus and dorsolateral subnuclei). They appeared to comprise several morphological types, but formed mainly asymmetrical synapses, most often with dendrites of varying size, and contained spherical clear vesicles together with fewer dense-cored vesicles. Substance P- and neurokinin A-immunoreactive terminals were fewer in number (9% of all terminals) but similar in appearance, with the immunoreaction restricted to the dense-cored vesicles. Analysis of serial- and double-labelled sections showed a co-existence of substance P and neurokinin A-immunoreactivity in 21% of glutamate-immunoreactive terminals. Immunoreactivity for gamma-aminobutyric acid was found in 33% of all terminals in the nucleus tractus solitarii. These predominantly contained pleomorphic vesicles and formed symmetrical synapses on dendrites and somata. Possible sites of axo-axonic contact by gamma-aminobutyric acid-immunoreactive terminals onto glutamate-or tachykinin-immunoreactive terminals were rare, but examples of adjacent glutamate and gamma-aminobutyric acid-immunoreactive terminals synapsing on the same dendritic profile were frequent. These results provide an anatomical basis for a gamma-aminobutyric acid mediated inhibition of glutamatergic excitatory inputs to the nucleus tractus solitarii at a post-synaptic level.     

Comparison of desflurane with isoflurane or propofol in spontaneously breathing ambulatory patients

The coexistence of S100beta with calcitonin gene-related peptide (CGRP), substance P (SP), somatostatin (SOM), nicotinamide adenosine dinucleotide phosphate-diaphorase (NADPH-d), and tyrosine hydroxylase (TH) was examined in the glossopharyngeal and vagal sensory ganglia. S100beta immunoreactive (-ir) neurons in the jugular and petrosal ganglia frequently colocalized CGRP- or SP-ir, whereas S100beta-ir neurons in the nodose ganglion infrequently contained CGRP- or SP-ir. No S100beta-ir neurons in the jugular and petrosal ganglia showed SOM-ir while the small number of SOM-ir neurons in the nodose ganglion colocalized S100beta-ir. Many neurons in the nodose ganglion colocalized S100beta-ir and NADPH-d activity, whereas S100beta-ir neurons in the jugular and nodose ganglia infrequently contained NADPH-d activity. S100beta- and TH-ir were frequently colocalized in nodose ganglion but not in petrosal or jugular ganglion neurons. These findings suggest relationships between S100beta and specific putative transmitters in functions of subpopulations of vagal and glossopharyngeal sensory neurons.     

Substrate assistance in the mechanism of family 18 chitinases: theoretical studies of potential intermediates and inhibitors

Gastric adaptive relaxation is a vago-vagal reflex, probably involving the site of interface of vagal afferents and efferents in the dorsal vagal complex of the medulla. Previous studies have shown that both substance P and nitric oxide in the dorsal vagal complex decrease intragastric pressure. The purpose of this study is, firstly, to localize NK1 tachykinin receptor immunoreactive (ir) staining in the dorsal vagal complex and, secondly, to determine its anatomical relationship to nitrergic cells in the dorsal motor nucleus of the vagus. Sections were stained by avidin-biotin immunocytochemistry using antiserum to NK1 receptor alone or combined with NADPH-diaphorase histochemistry. In the nucleus tractus solitarius, NK1 receptor-ir varicosities were moderately dense in the medial subnucleus, but sparse in the centralis and gelatinosus subnuclei. In the dorsal motor nucleus of the vagus, NK1 receptor-ir staining in cell bodies and fibers was present throughout, with a markedly dense varicose fiber and cell body staining in a lateral column of the rostral portion of the nucleus. NADPH-diaphorase staining is most marked in cell bodies in the same region of the dorsal motor nucleus of the vagus. In dual-stained sections, there was complete overlap of NADPH-diaphorase and NK1 receptor-ir stain; however, the markers were very rarely colocalized within the same vagal motor neurons. Ipsilateral vagotomy almost completely abolished NK1r-ir staining in vagal motor neurons. We conclude that, in the dorsal motor nucleus of the vagus, NK1 receptor is synthesized by a population of vagal motor neurons which are in close anatomical proximity to, but separate from, nitrergic neurons. Based on these observations, substance P-mediated gastric relaxation in this region is unlikely to be via activation of nitrergic vagal preganglionic neurons. In the nucleus tractus solitarius, the NK1 receptor and NADPH-diaphorase stain are not codistributed in subnuclei mediating gastric and esophageal control. Therefore, substance P and nitric oxide may mediate their respective gastrointestinal effects via separate afferent pathways.     

Vagal and spinal afferent innervation of the rat esophagus: a combined retrograde tracing and immunocytochemical study with special emphasis on calcium-binding proteins

Vagal afferent neurons contain a variety of neurochemical markers and neuroactive substances, most of which are present also in dorsal root ganglion cells. To test for the suitability of the calcium-binding protein calretinin as a specific marker for vagal afferent fibers in the periphery, immunocytochemistry for this protein was combined with retrograde tracing. Nerve fibers in the rat esophagus, as well as vagal and spinal sensory neurons innervating the esophagus, were investigated for co-localization of calretinin with calbindin, calcitonin gene-related peptide, and NADPH diaphorase. The results indicated that calretinin immunocytochemistry demonstrates neuronal structures known as vagal afferent from other studies, in particular intraganglionic laminar endings. A few enteric neurons whose distribution was unrelated to intraganglionic laminar endings also stained for calretinin. Strikingly, calretinin immunoreactivity was absent from spinal afferent neurons innervating the rat esophagus. In intraganglionic laminar endings and nodose ganglion cells calretinin was highly co-localized with calbindin but not with calcitonin gene-related peptide. On the other hand, calbindin was also found in spinal afferents to the esophagus where it was co-localized with calcitonin gene-related peptide. Vagal afferent neurons innervating the esophagus were never positive for NADPH diaphorase. Thus, calretinin appears to be a more specific marker for vagal afferent structures in the esophagus than calbindin, which is expressed by both vagal and spinal sensory neurons. Calretinin immunocytochemistry may be utilized as a valuable tool for investigations of subpopulations of vagal afferents in certain viscera.     

Neural control of left ventricular contractility in the dog heart: synaptic interactions of negative inotropic vagal preganglionic neurons in the nucleus ambiguus with tyrosine hydroxylase immunoreactive terminals

Recent physiological evidence indicates that vagal postganglionic control of left ventricular contractility is mediated by neurons found in a ventricular epicardial fat pad ganglion. In the dog this region has been referred to as the cranial medial ventricular (CMV) ganglion [J.L. Ardell, Structure and function of mammalian intrinsic cardiac neurons, in: J.A. Armour, J.L. Ardell (Eds.). Neurocardiology, Oxford Univ. Press, New York, 1994, pp. 95-114; B.X. Yuan, J.L. Ardell, D.A. Hopkins, A.M. Losier, J.A. Armour, Gross and microscopic anatomy of the canine intrinsic cardiac nervous system, Anat. Rec., 239 (1994) 75-87]. Since activation of the vagal neuronal input to the CMV ganglion reduces left ventricular contractility without influencing cardiac rate or AV conduction, this ganglion contains a functionally selective pool of negative inotropic parasympathetic postganglionic neurons. In the present report we have defined the light microscopic distribution of preganglionic negative inotropic neurons in the CNS which are retrogradely labeled from the CMV ganglion. Some tissues were also processed for the simultaneous immunocytochemical visualization of tyrosine hydroxylase (TH: a marker for catecholaminergic neurons) and examined with both light microscopic and electron microscopic methods. Histochemically visualized neurons were observed in a long slender column in the ventrolateral nucleus ambiguus (NA-VL). The greatest number of retrogradely labeled neurons were observed just rostral to the level of the area postrema. TH perikarya and dendrites were commonly observed interspersed with vagal motoneurons in the NA-VL. TH nerve terminals formed axo-dendritic synapses upon negative inotropic vagal motoneurons, however the origin of these terminals remains to be determined. We conclude that synaptic interactions exist which would permit the parasympathetic preganglionic vagal control of left ventricular contractility to be modulated monosynaptically by catecholaminergic afferents to the NA-VL.     

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.     

The subthalamic nucleus and the external pallidum: two tightly interconnected structures that control the output of the basal ganglia in the monkey

The aim of the present study was to elucidate the organization of the interconnections between the subthalamic nucleus and the two segments of the globus pallidus in squirrel monkeys. By making small deposits of tracers in the two segments of the globus pallidus, we demonstrate that interconnected neurons of the subthalamic nucleus and the external pallidum innervate, via axon collaterals, the same population of neurons in the internal pallidum. Furthermore, this organizational principle holds true for different functional regions of the pallidum and the subthalamic nucleus. Injections of biotinylated dextran amine were made in the dorsal (associative), ventrolateral (sensorimotor) and rostromedial (limbic) regions of the internal pallidum. Following these injections, there were rich clusters of labelled terminals in register with retrogradely labelled perikarya in related functional regions of the subthalamic nucleus and the external pallidum. At the electron microscopic level, the majority of labelled terminals in the external pallidum displayed the ultrastructural features of boutons from the subthalamic nucleus and were non-immunoreactive for GABA, whereas those in the subthalamic nucleus resembled terminals from the external pallidum and displayed GABA immunoreactivity. In both cases, the synaptic targets of the labelled terminals included labelled neurons. These observations suggest that the biotinylated dextran amine injected in the internal globus pallidus was transported retrogradely to perikarya in the external pallidum and the subthalamic nucleus and then anterogradely, via axon collaterals, to the subthalamic nucleus and the external pallidum respectively. This suggestion was supported by injections of biotinylated dextran amine or Phaseolus vulgaris-leucoagglutinin in regions of the external pallidum that corresponded to those containing retrogradely labelled cells following injections in the internal pallidum. The clusters of labelled cells and varicosities that resulted from these injections were found in regions of the subthalamic nucleus similar to those labelled following injections in the internal globus pallidus. Furthermore, terminals from the external pallidum and the subthalamic nucleus converged on the same regions in the internal globus pallidus. The results of the present tracing study define the basic network underlying the interconnections between the external segment of the globus pallidus and the subthalamic nucleus, and their connections with the output neurons of the basal ganglia in primates.     

Selective effect of 2-(polyhydroxyalkyl)-thiazolidine-4-carboxylic acids on nonprotein sulfhydryl groups in tumor bearing mice

Several substances have been reported as candidates for the neurotransmitter in the laryngeal afferent system. In the present study we demonstrated that catecholamine is also a candidate neurotransmitter in the canine laryngeal afferent system using tyrosine hydroxylase (TH) immunochemistry in combination with retrograde labelling with cholera toxin B in subunit-conjugated gold (CTBG). A few cells in the nodose ganglion labelled by application of CTBG to the internal branch of the superior laryngeal nerve were also TH-immunoreactive. These cells were also labelled following application of CTBG to the nucleus of the solitary tract. These results indicate that some of the TH-IR cells in the nodose ganglion could be primary afferent neurones for the canine larynx.     

Selective stimulation of jugular ganglion afferent neurons in guinea pig airways by hypertonic saline

We evaluated the ability of hyperosmolar stimuli to activate afferent nerves in the guinea pig trachea and main bronchi and investigated the neural pathways involved. By using electrophysiological techniques, studies in vitro examined the effect of hyperosmolar solutions of sodium chloride (hypertonic saline) on guinea pig airway afferent nerve endings arising from either vagal nodose or jugular ganglia. The data reveal a differential sensitivity of airway afferent neurons to activation with hypertonic saline. Afferent fibers (both A delta and C fibers) with cell bodies located in jugular ganglia were much more sensitive to stimulation with hypertonic saline, compared with afferent neurons with cell bodies located in nodose ganglia. Additional studies in vivo demonstrated that inhalation of aerosols of hypertonic saline induced plasma extravasation in guinea pig trachea that was mediated via tachykinin NK1 receptors. Identification of a differential sensitivity of guinea pig airway afferent nerves to hypertonic saline leads to the speculation that airway responses to hyperosmolar stimuli may result from activation of afferent neurons originating predominantly from the jugular ganglion.     

Localization of allatostatin-immunoreactive material in the central nervous system, stomatogastric nervous system, and gut of the cockroach Blattella germanica

Immunoreactivity against peptides of the allatostatin family having a typical YXFGL-NH2 C-terminus has been localized in different areas of the central nervous system, stomatogastric nervous system and gut of the cockroach Blattella germanica. In the protocerebrum, the most characteristic immunoreactive perikarya are situated in the lateral and median neurosecretory cell groups. Immunoreactive median neurosecretory cells send their axons around the circumesophageal connectives to form arborizations in the anterior neuropil of the tritocerebrum. A group of cells in the lateral aspect of the tritocerebrum project to the antennal lobes in the deutocerebrum, where immunoreactive arborizations can be seen in the periphery of individual glomeruli. Nerve terminals were shown in the corpora allata. These terminals come from perikarya situated in the lateral neurosecretory cells in the pars lateralis and in the subesophageal ganglion. Immunoreactive axons from median neurosecretory cells and from cells positioned in the anteriormost part of the tritocerebrum enter together in the stomatogastric nervous system and innervate foregut and midgut, especially the crop and the valve between the crop and the midgut. The hindgut is innervated by neurons whose perikarya are located in the last abdominal ganglion. Besides immunoreactivity in neurons, allatostatin-immunoreactive material is present in endocrine cells distributed within the whole midgut epithelium. Possible functions for these peptides according to their localization 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.     

Capsaicin-resistant vagal afferent fibers in the rat gastrointestinal tract: anatomical identification and functional integrity

The presence and distribution of vagal fibers and terminals throughout esophagus and gastrointestinal tract that could be anterogradely labeled by nodose ganglion tracer injections was quantitatively assessed in capsaicin- and vehicle-pretreated adult rats, in order to identify the capsaicin-resistant population. Up to 90% of the intraganglionic laminar endings (IGLEs), in the myenteric plexus of the esophagus, and 70-90% in the stomach, as well as 57% of the intramuscular endings or arrays (IMAs) in the fundic stomach survived the capsaicin treatment, while in the upper small intestine only few and in the lower small intestine, the cecum and colon, virtually no IGLEs survived capsaicin treatment. Intramucosal terminals were not assessed. Furthermore, gastric balloon distension-induced c-Fos expression in the dorsal vagal complex was not significantly decreased in capsaicin-treated rats. It is concluded that among primary vagal afferents there is a capsaicin-resistant population that primarily innervates the esophagus and upper gastrointestinal tract, and a capsaicin-sensitive population that innervates mainly the lower tract. At least vagal gastric tension-sensitive afferents also seems to be functionally intact in that they may be capable of synaptically activating second-order neurons in the brainstem.     

The arcuate nucleus is the major source for neuropeptide Y-innervation of thyrotropin-releasing hormone neurons in the hypothalamic paraventricular nucleus

Neuropeptide Y (NPY) immunoreactive (-ir) nerve fibers densely innervate hypophysiotropic TRH perikarya and dendrites in the hypothalamic paraventricular nucleus (PVN). To evaluate the contribution of the arcuate nucleus (Arc) to this innervation, the effect of Arc ablation by neonatal monosodium glutamate (MSG) treatment on the density of NPY-fibers contacting TRH neurons in the PVN was investigated. After the lesioned animals and vehicle-treated controls reached adulthood, the number of contacts between NPY-ir boutons and TRH-ir perikarya in the PVN was determined in double-immunostained sections. In controls, numerous contacts between NPY-ir terminals and TRH perikarya and dendrites were observed, confirming earlier findings. MSG treatment resulted in a marked reduction of the size of the Arc and also the number of NPY-perikarya with a concomitant reduction of 82.4 +/-2.1% in the relative number of NPY terminals contacting TRH perikarya and first order dendrites in the medial parvocellular and periventricular subdivisions of the PVN. In contrast, lesioning of the ascending adrenergic bundle in the brain stem caused no statistically significant change in the number of NPY-terminals in close apposition to hypophysiotropic TRH neurons in the PVN. These data confirm earlier findings that NPY-containing axon terminals innervate TRH neurons in the PVN and further demonstrate a potentially important anatomical relationship between NPY-producing neurons in the Arc and hypophysiotropic TRH neurons.     

The regulation mechanism of HLA class II gene expression at the level of mRNA stability

Sensory ganglia (trigeminal, jugular, nodose, cervical and lumbar dorsal root ganglia) of the guinea-pig were investigated for the presence of a constitutive carbon monoxide-generating enzyme, heme oxygenase-2 (HO-2). A 36-kDa HO-2 immunoreactive protein was identified by Western blotting in protein extracts from dorsal root ganglia and localized by immunohistochemistry to all neuronal perikarya, including both substance P-positive and substance P-negative neurons, in all ganglia investigated. This ubiquitous distribution points to a general requirement for HO-2 in primary afferent neurons rather than to an association with a specific functionally defined subpopulation. Neither the axons of the sensory neurons nor their peripheral terminals in the skin and around visceral arteries were HO-2 immunoreactive. Explants of dorsal root ganglia with crushes placed on the dorsal roots showed accumulation of the neuropeptide, substance P, at the ganglionic side of the crush, but these axons were non-reactive to HO-2, indicating that there is no substantial transport of HO-2 towards the central ending of these sensory neurons. Collectively, the findings suggest that HO-2 exerts it major effects within the sensory ganglia themselves.     

Nitric oxide-synthesising neurons in the central subnucleus of the nucleus tractus solitarius provide a major innervation of the rostral nucleus ambiguus in the rabbit

We describe an intramedullary nitric oxide synthase (NOS) neural pathway that projects from the nucleus tractus solitarius (NTS) to the rostral nucleus ambiguus (NA) in the rabbit. With the use of NADPH diaphorase histochemistry and NOS immunohistochemistry, a compact group of NOS-positive perikarya was identified in the central subnucleus of the NTS dorsomedial to the tractus solitarius and rostral to the obex. A dense network of NOS terminals was seen in the rostral NA. We investigated whether NOS terminals in the NA derive from NOS perikarya in the central NTS and whether the central NOS pathway links esophageal afferents and efferents. In some rabbits, the central NTS was unilaterally lesioned. In others, Phaseolus vulgaris-leucoagglutinin (PHA-L) was injected into the central NTS, or cholera toxin-gold was injected into the NA, or cholera toxin-horseradish peroxidase (HRP) was injected into the wall of the esophagus. The medulla was subsequently processed to demonstrate PHA-L, cholera toxin-gold, HRP, and NOS reactivity. Seven days after the NTS lesion, we observed a marked decrease in the density of NOS terminals in the ipsilateral NA. After injection of PHA-L into the central NTS, a dense group of PHA-L fibres was seen in the rostral NA, principally ipsilaterally. Afferent fibres from the esophagus were found around the NOS cell bodies in the central NTS, and many of these NOS neurons were double labeled with cholera toxin-gold after injection of this tracer into the NA. NOS terminals were found around NA neurons that were retrogradely labelled from the esophagus. We conclude that the NOS neurons in the central NTS act as interneurons in a central pathway connecting esophageal afferents and efferents.