Ultrastructure of reduced nicotinamide adenine dinucleotide phosphate (NADPH) diaphorase-positive neurons in the cat cerebral cortex, amygdala and caudate nucleus

The ultrastructure of reduced nicotinamide adenine dinucleotide phosphate (NADPH) diaphorase-positive neurons in cat cerebral cortex, amygdala and caudate nucleus was investigated by electron microscopy using a modified method applicable to aldehyde-fixed tissues. These NADPH diaphorase-positive neurons were morphologically similar to neurons immunohistochemically positive for somatostatin. They had large amounts of electron-dense formazan reaction products scattered through the whole cytoplasm but not in the mitochondria or nucleus. Similar electron-dense reaction products were visible in the dendrites of these neurons. The results indicate that NADPH diaphorase histochemistry is a useful method for the ultrastructural examination of particular groups of neurons.     

Synaptic interaction of vagal afferents and catecholaminergic neurons in the rat nucleus tractus solitarius

Combined radioautography and immunocytochemistry were used to define the ultrastructure and synaptic relations between vagal sensory afferents and catecholaminergic (CA) neurons of the A2 group located within the nucleus tractus solitarius (NTS) of rat brain. The vagal afferents were radioautographically labeled by tritiated amino acids anterogradely transported from the nodose ganglion. Immunocytochemical labeling for tyrosine hydroxylase (TH) served for the identification of catecholaminergic neurons. The radiographically labeled axons seen by light microscopy were widely distributed throughout the more caudal NTS. The reduced silver grains were more densely distributed within the NTS located homolateral to the injected nodose ganglion. The radioautographically labeled processes were localized in regions containing catecholaminergic neurons as indicated by immunoreactivity for TH. Electron microscopic analysis of the medial NTS at the level of the obex demonstrated that the reduced silver grains were localized within axon terminals. The radioautographically labeled terminals were 2-3 microns in diameter, contained numerous small, clear and a few large, dense vesicles, and formed predominately axodendritic synapses. Many of the recipient dendrites contained immunoreactivity for TH. In rare instances, vagal afferents formed synaptic appositions with both TH-labeled and unlabeled axon terminals and neuronal soma. This study provides the first ultrastructural evidence that the catecholaminergic neurons within the NTS receive direct synapses from sensory neurons in the nodose ganglion.     

Morphological analysis of vagal input to gastrin releasing peptide and vasoactive intestinal peptide containing neurons in the rat glandular stomach

Vagal preganglionic efferents to the rat stomach were labeled anterogradely by injecting the fluorescent carbocyanine dye DiA into the dorsal motor nucleus in vivo. Enteric neurons were labeled in toto by intraperitoneal administration of Fluorogold, and neurochemically characterized by simultaneous single- and double-label immunocytochemistry. Single peptide immunocytochemistry revealed that in all three major areas of the stomach, about one-third of all gastrin-releasing peptide immunoreactive (GRP-IR) neurons in the myenteric plexus, received vagal contacts. Because the proportion of GRP-IR neurons was 32% in the fundus, 23% in the corpus, and only 8% in the antrum, the absolute number of vagally contacted GRP-IR neurons per cm² was also different. Double-label immunocytochemistry revealed colocalization of vasoactive intestinal peptide immunoreactivity (VIP-IR) in 45%, and of enkephalin immunoreactivity (ENK-IR) in about 30%, of the GRP-IR myenteric neurons. A subpopulation of myenteric neurons colocalized GRP-IR and VIP-IR and projects almost exclusively to the gastrin cell-rich basal mucosa of the antrum and the oxyntic mucosa of the corpus. Another subpopulation containing GRP-IR, but not VIP-IR, projects mainly to the myenteric plexus itself and the external muscle layers, particularly the longitudinal muscle. A third group of neurons containing VIP-IR but not GRP-IR projects heavily to the circular muscle layer, the muscularis mucosae, and to other myenteric neurons. Vagal input to these three subpopulations seems not to be selective, in that an equal proportion of about 20 to 30% of each group was vagally contacted. Vagal inputs to these neurochemically and topographically distinct enteric neurons provide the basis for the physiological vagal control of gastrin release, gastric acid secretion, and gastric motility. © 1996 Wiley-Liss, Inc.     

Descending inhibition of spinal neurons in the cardiopulmonary region by electrical stimulation of vagal afferent nerves

Neurons in the left upper thoracic spinal cord of cats anesthetized with chloralose and relaxed with succinylcholine were activated by electrical stimulation of sympathetic chains or by pinching of the skin. Most neurons were spontaneously active, and 22 of the 51 neurons studied were antidromically activated from the spinomedullary junction, demonstrating their projection to higher centers. Stimulation of the central cut ends of either cervical vagus resulted in inhibition of firing in 26 neurons, acceleration in 6 neurons, both effects in 2 neurons, and no effect in the remaining 17 neurons. The parameters of stimulation determined whether inhibition or facilitation was produced in the two neurons that showed either effect. Two spinal neurons were fired by vagal stimulation with latencies of 16 and 20 msec. Activity in vagal afferent fibers is believed to impinge upon brain stem structures that have been shown by others to cause descending inhibition (and facilitation) of spinothalamic and spinoreticular neurons. This would provide a mechanism for modulation of sensory information from the cardiopulmonary region.     

Immunocytochemical characterization of rat brainstem neurons with vagal afferent input from the stomach challenged by acid or ammonia

Exposure of the gastric mucosa to backdiffusing acid is signalled to the brainstem via vagal afferents. This study examined whether exposure of the Sprague-Dawley rat stomach to hydrochloric acid (HCl) or ammonium hydroxide (NH4OH), a noxious chemical produced by Helicobacter pylori, activates different vagal afferent pathways as reflected by different circuitries in the medullary brainstem. Two hours after intragastric treatment with HCl or NH4OH the activation of neurons in the nucleus tractus solitarii at the rostrocaudal extension of the area postrema (NTSAP) was visualized by c-Fos immunohistochemistry and their chemical coding characterized by double-labelling immunohistochemistry. Exposure of the rat gastric mucosa to HCl (0.15-0.5 M) or NH4OH (0.1-0.3 M) led to a concentration-dependent expression of c-Fos in the NTSAP. The number and distribution of NTSAP neurons activated by 0.35 M HCl and 0.3 M NH4OH were similar; the highest number of activated neurons occurring in the medial part of the NTSAP. Some 60% of the NTSAP neurons activated by intragastric HCl and NH4OH stained for the high affinity glutamate transporter EAAC1, while some 30% contained calbindin or neuropeptide Y. Glutamate receptors of the N-methyl-D-aspartate type were found on approximately 50% of the c-Fos-positive cells in the NTSAP, whereas tachykinin NK1, NK2 and NK3 receptors were present on 5-10% of the activated neurons. The similar number and distribution of c-Fos-expressing neurons within the NTSAP and their identical chemical coding indicate that exposure of the rat stomach to backdiffusing concentrations of HCl and NH4OH activates the same vagal afferent-NTSAP pathway.     

Nitric oxide inhibits excitatory vagal afferent input to nucleus tractus solitarius neurons in anaesthetized rats

Objective

Endogenous nitric oxide (NO) has been implicated in the regulation of neuronal activity which mediates cardiovascular reflexes. However, there is controversy concerning the role of NO in the nucleus tractus solitarius (NTS). The present study aims to elucidate the possible physiological role of endogenous NO in modulating the excitatory vagal afferent input to NTS neurons.

Methods

All the experiments in the rat were conducted under anaesthetic conditions. Ionophoresis method was used for the application of NO donor or nitric oxide synthase (NOS) inhibitor, and single unit recording method was employed to detect the effects of these applications on vagal afferent- or cardio-pulmonary C-fibre reflex-evoked neuronal excitation in NTS.

Results

Ionophoresis applications of L-arginine (L-Arg), a substrate of NOS, and sodium nitroprusside (SNP), a NO donor, both attenuated the vagal afferent-evoked discharge by (51.5±7.6)% (n = 17) and (68.3±7.1)% (n = 9), respectively. In contrast, application of D-Arg at the same current exerted no overall effect on this input. Also, both L-Arg and SNP inhibited spontaneous firing of most of the recorded neurons. In contrast, ionophoresis application of NG-nitro-Larginine methyl ester (L-NAME) enhanced vagal afferent-evoked excitation by (66.3±11.4)% (n = 7). In addition, ionophoresis application of L-Arg and SNP significantly attenuated cardio-pulmonary C-fibre reflex-induced excitation in the tested NTS neurons.

Conclusion

Activation of local NO pathway in the NTS could suppress vagal afferent-evoked excitation, suggesting that NO is an important neuromodulator of visceral sensory input in the NTS.     

N-methyl-D-aspartate receptor subunit phenotypes of vagal afferent neurons in nodose ganglia of the rat

Most vagal afferent neurons in rat nodose ganglia express mRNA coding for the NR1 subunit of the heteromeric N-methyl-D-aspartate (NMDA) receptor ion channel. NMDA receptor subunit immunoreactivity has been detected on axon terminals of vagal afferents in the dorsal hindbrain, suggesting a role for presynaptic NMDA receptors in viscerosensory function. Although NMDA receptor subunits (NR1, NR2B, NR2C, and NR2D) have been linked to distinct neuronal populations in the brain, the NMDA receptor subunit phenotype of vagal afferent neurons has not been determined. Therefore, we examined NMDA receptor subunit (NR1, NR2B, NR2C, and NR2D) immunoreactivity in vagal afferent neurons. We found that, although the left nodose contained significantly more neurons (7,603), than the right (5,978), the proportions of NMDA subunits expressed in the left and right nodose ganglia were not significantly different. Immunoreactivity for NMDA NR1 subunit was present in 92.3% of all nodose neurons. NR2B immunoreactivity was present in 56.7% of neurons; NR2C-expressing nodose neurons made up 49.4% of the total population; NR2D subunit immunoreactivity was observed in just 13.5% of all nodose neurons. Double labeling revealed that 30.2% of nodose neurons expressed immunoreactivity to both NR2B and NR2C, whereas NR2B and NR2D immunoreactivities were colocalized in 11.5% of nodose neurons. NR2C immunoreactivity colocalized with NR2D in 13.1% of nodose neurons. Our results indicate that most vagal afferent neurons express NMDA receptor ion channels composed of NR1, NR2B, and NR2C subunits and that a minority phenotype that expresses NR2D also expresses NR1, NR2B, and NR2C.     

Vagal and splanchnic effects at the level of the ventromedian nucleus of the hypothalamus (VMH) in the cat

A systematic study of vagal and splanchnic projections to the VMH area was undertaken in anaesthetized cats by means of macroelectrodes and microelectrodes. Responses elicited by vagal and splanchnic nerve stimulation were recorded from the VMH nucleus and the region situated above it. The fact that they are identical, whatever the nerve stimulated, indicates that the afferences follow a common central pathway. From the response latency and the stimulation parameters, it is assumed that the small vagal and splanchnic fibres (Aδ, B and C) are involved. Evoked potentials are generally constituted of two parts: the earlier implies certainly the VPL nucleus (since it disappears definitively after VPL coagulation), whereas the later concerns a different relay probably located in the neighbouring and associative structure (since it persists after VPL coagulation). Similar late responses were simultaneously recorded in VPL nucleus, suggesting that this structure was the same for both nuclei. Its exact location was discussed.On the other hand, effects of gastric distension on the evoked potentials produced by vagal and splanchnic nerve stimulation were studied. The results obtained (decrease or increase in the responses, occurrence of an additional potential) indicated clearly that the gastric afferences projected to the VMH region. This simple method could be used to determine the importance of the sensory innervation of the different parts of the digestive tract.     

Neurons co-localizing calretinin immunoreactivity and reduced nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-d) activity in the hippocampus and dentate gyrus of the rat

Co-localization of calretinin immunoreactivity and nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-d) activity was studied in the rat hippocampus and dentate gyrus. Neurons co-expressing both markers (CR/NADPH-d) were observed throughout the hippocampus and dentate gyrus. However, they were more abundant in the stratum pyramidale and radiatum of CA3, stratum pyramidale of CA1, and in the juxtagranular zone of the hilus. The NADPH-d activity appeared in 37% of the calretinin immunoreactive neurons in CA3, 42% in CA1, and 36% in the dentate gyrus, whereas calretinin immunoreactivity occurred in 41% of the NADPH-d positive neurons in the hippocampus, and 16% in the dentate gyrus. The morphology and location of the double marked cells could not be used as a characteristic of the co-localizing neurons. The heavily stained NADPH-d neurons occurring mainly in CA1 do not show calretinin immunoreactivity. NADPH-d fiber swellings could be observed in close apposition to calretinin immunoreactive neurons and dendrites, suggesting synaptic contacts. It has been reported that calretinin immunoreactivity and NADPH-d activity co-localize infrequently in other areas such as the neocortex, striatum, hypothalamus and tegmental nucleus. The relatively high proportion of double marked cells found in the hippocampus and dentate gyrus could be indicative of the importance of the CR/NADPH-d interneurons in the circuitries of these areas.     

Coexistence of calbindin D-28k and NADPH-diaphorase in vagal and glossopharyngeal sensory neurons of the rat

The presence and coexistence of calbindin D-28k-immunoreactivity (ir) and nicotinamide adenosine dinucleotide phosphate (NADPH)-diaphorase activity (a marker of neurons that are presumed to convert L-arginine to L-citrulline and nitric oxide) were examined in the glossopharyngeal and vagal sensory ganglia (jugular, petrosal and nodose ganglia) of the rat. Calbindin D-28k-ir nerve cells were found in moderate and large numbers in the petrosal and nodose ganglia, respectively. Some calbindin D-28k-ir nerve cells were also observed in the jugular ganglion. NADPH-diaphorase positive nerve cells were localized to the jugular and nodose ganglia and were rare in the petrosal ganglion. A considerable portion (33–51%) of the NADPH-diaphorase positive neurons in these ganglia colocalized calbindin D-28k-ir. The presence and colocalization of calbindin D-28k-ir and NADPH-diaphorase activity in neurotransmitter-identified subpopulations of visceral sensory neurons were also studied. In all three ganglia, calcitonin gene-related peptide (CGRP)-ir was present in many NADPH-diaphorase positive neurons, a subset of which also contained calbindin D-28k-ir. In the nodose ganglion, many (42%) of tyrosine hydroxylase (TH)-ir neurons also contained NADPH diaphorase activity but did not contain calbindin D-28k-ir. These data are consistent with a potential co-operative role for calbindin D-28k and NADPH-diaphorase in the functions of a subpopulation of vagal and glossopharyngeal sensory neurons.     

Distribution of reduced nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-d) cells and fibers in the monkey amygdaloid complex.

The NADPH-d histochemical method stains a selective population of neurons in the central nervous system. Although the functional significance of the enzyme in these cells is unknown, it has nonetheless proved to be a useful marker. In the present study we describe the distribution of NADPH-d-positive cells and fibers in the amygdaloid complex of the Macaca fascicularis monkey. NADPH-d-positive neurons were distributed throughout the amygdaloid complex. Based on the intensity of the reaction product, three different types of NADPH-d-positive cells were described: type 1 cells, the most intensely stained, varied in morphology and were most commonly found in the accessory basal, basal, and lateral nuclei and in the nucleus of the lateral olfactory tract; type 2 cells, the most common NADPH-d-positive cells, were more lightly stained, were generally stellate in shape, and were found in the lateral, basal, and accessory basal nuclei; type 3 cells were very lightly stained, oval or round in shape, and mostly found in the medial, anterior cortical, and paralaminar nuclei. NADPH-d staining was also associated with axonal fiber plexuses in various regions of the amygdala. The highest densities of stained fibers were found in the lateral nucleus, the parvicellular portion of the accessory basal nucleus, and the anterior amygdaloid area. The lowest densities of NADPH-d-positive fiber staining were found in the amygdalohippocampal area, in the lateral part of the central nucleus, and in the intercalated nuclei. In addition to the neuronal and fiber staining, a diffuse, blue neuropil staining was also observed, most commonly in the anterior cortical nucleus, the medial nucleus, the intercalated nuclei, and especially in the amygdalohippocampal area. The distribution of NADPH-d staining often respected nuclear boundaries within the amygdala and was particularly helpful in clarifying the borders of the amygdalohippocampal area.     

Coexistence of S100β and putative transmitter agents in vagal and glossopharyngeal sensory neurons of the rat

The coexistence of S100β 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. S100β immunoreactive (-ir) neurons in the jugular and petrosal ganglia frequently colocalized CGRP- or SP-ir, whereas S100β-ir neurons in the nodose ganglion infrequently contained CGRP- or SP-ir. No S100β-ir neurons in the jugular and petrosal ganglia showed SOM-ir while the small number of SOM-ir neurons in the nodose ganglion colocalized S100β-ir. Many neurons in the nodose ganglion colocalized S100β-ir and NADPH-d activity, whereas S100β-ir neurons in the jugular and nodose ganglia infrequently contained NADPH-d activity. S100β- and TH-ir were frequently colocalized in nodose ganglion but not in petrosal or jugular ganglion neurons. These findings suggest relationships between S100β and specific putative transmitters in functions of subpopulations of vagal and glossopharyngeal sensory neurons.     

A comparative cluster analysis of nicotinamide adenine dinucleotide phosphate (NADPH)‐diaphorase histochemistry in the brains of amphibians

Nicotinamide adenine dinucleotide phosphate–diaphorase (NADPH‐d) is a key enzyme in the synthesis of the gaseous neurotransmitter nitric oxide. We compare the distribution of NADPH‐d in the brain of four species of hylid frogs. NADPH‐d–positive fibers are present throughout much of the brain, whereas stained cell groups are distributed in well‐defined regions. Whereas most brain areas consistently show positive neurons in all species, in some areas species‐specific differences occur. We analyzed our data and those available for other amphibian species to build a matrix on NADPH‐d brain distribution for a multivariate analysis. Brain dissimilarities were quantified by using the Jaccard index in a hierarchical clustering procedure. The whole brain dendrogram was compared with that of its main subdivisions by applying the Fowlkes–Mallows index for dendrogram similarity, followed by bootstrap replications and a permutation test. Despite the differences in the distribution map of the NADPH‐d system among species, cluster analysis of data from the whole brain and hindbrain faithfully reflected the evolutionary history (framework) of amphibians. Dendrograms from the secondary prosencephalon, diencephalon, mesencephalon, and isthmus showed some deviation from the main scheme. Thus, the present analysis supports the major evolutionary stability of the hindbrain. We provide evidence that the NADPH‐d system in main brain subdivisions should be cautiously approached for comparative purposes because specific adaptations of a single species could occur and may affect the NADPH‐d distribution pattern in a brain subdivision. The minor differences in staining pattern of particular subdivisions apparently do not affect the general patterns of staining across species. J. Comp. Neurol. 522:2980–3003, 2014. © 2014 Wiley Periodicals, Inc.     

Synaptic connections of neuropeptide Y (NPY) immunoreactive neurons in the hilar area of the rat hippocampus

Synaptic connections and fine structural characteristics of neuropeptide Y-immunoreactive (NPY-i) neurons in the fascia dentata were studied using an antiserum against NPY. Normal and colchicine pretreated rats were examined to study the synaptic connections of NPY-i neurons in the normal fascia dentata. The perforant pathway and fimbria fornix were transected to label afferent fibers to NPY-positive cells. Horseradish peroxidase conjugated with wheat germ agglutinin (HRP-WGA) was injected into the contralateral hippocampus to study commissural projections of hippocampal NPY-i neurons, and to search for NPY-i synaptic contacts on immunonegative commissural cells. Since earlier reports have shown that at least half of the NPY-i neurons also contain somatostatin (SS), the distribution of NPY-i neurons in the hilar area was determined and compared with that of SS-i neurons. Four types of dentate NPY-i neurons were distinguished: Type 1: large multipolar cells in the deep hilus (9%). Type 2: medium-sized multipolar and fusiform hilar neurons with dendrites occasionally reaching the outer molecular layer (64%). Type 3: pyramidal shaped cells in the granule cell layer with long apical dendrites reaching the outer molecular layer (20%). Type 4: small multipolar NPY-i cells located in the molecular layer (7%). Our results indicate two overlapping but not identical cell populations of NPY-i and SS-i neurons. Light and electron microscopic analysis of the normal fascia dentata demonstrated that the majority of NPY-i terminals are located in the outer molecular layer of the dentate gyrus, where they establish symmetric synaptic contacts on dendritic shafts and occasionally on spines of granule cells. A moderate number of NPY-i synapses were also found on dendrites in the inner molecular layer and on the cell body of granule cells. Numerous symmetric NPY-i synapses were found on dendrites and somata of neurons in the hilar area. Some NPY-i dendrites in the hilar area received mossy axon collateral input. After transection of the perforant pathway degenerated axon terminals could be found in synaptic contact with NPY-i dendrites in the outer molecular layer. Commissurotomy revealed direct commissural input to NPY-i dendrites in the inner molecular layer and in the hilus. After injection of HRP-WGA into the contralateral hippocampus 2% of hilar NPY-i neurons were retrogradely labeled and symmetric NPY-i synapses were found on the cell bodies and dendrites of unstained HRP-WGA labeled neurons.(ABSTRACT TRUNCATED AT 400 WORDS)     

Ultrastructural evidence of dopaminergic input to enkephalinergic neurons in rat neostriatum

The synaptic relationship between neuronal structures reacting with antibodies to tyrosine hydroxylase (TH) and Leu- or Met-enkephalin (ENK) was studied by the 'mirror technique' in adjacent sections of rat neostriatum. TH-immunoreactive (TH-IR) axonal boutons surrounding the neural perikarya and proximal dendrites of ENK-immunoreactive (ENK-IR) neurons were very thin (0.1-0.4 micrograms). They contained many small clear vesicles and sometimes had symmetrical membrane specializations. This provides morphological evidence for catecholaminergic, presumably dopaminergic inputs to rat striatal enkephalin neurons.     

Evidence of substance P immunoreactive neurons in dorsal root ganglia and vagal ganglia projecting to the guinea pig pylorus

The origin of extrinsic substance P fibers in the guinea pig pyloric wall was investigated by combining retrograde axonal tracing and indirect immunofluorescence techniques. After injection of Fast Blue into the pyloric wall labeled cells were found in the T7-T9 dorsal root ganglia and the nodose and jugular ganglia. About 60% of the labeled cells in the dorsal root ganglia contained substance P-like immunoreactivity. After local application of colchicine, a few substance P positive cells were observed in the nodose and jugular ganglia, some of which also contained Fast Blue.     

Effect of L-glutamic acid on acid secretion and immunohistochemical localization of glutamatergic neurons in the rat stomach

Glutamatergic neurons in the rat stomach were localized immunohistochemically using antibodies against L-glutamate (L-Glu) as well as glutamate synthesizing enzyme, glutaminase (GLNase). Myenteric ganglia and nerve bundles in the circular muscle and the longitudinal muscle were found to contain GLU- and GLNase-positive nerve fibers, while submucosa and mucosa were devoid of glutamatergic innervation. The distribution of glutamatergic neurons and their processes in both myenteric ganglia and circular muscle is heterogeneous within the stomach. The effect of L-Glu on gastric acid secretion was investigated on an everted preparation of isolated rat stomach. L-Glu at 10^?7 and 10^?8 M alone had no effect on acid secretion. It was found that the oxotremorine-, histamine-, or gastrin-stimulated acid secretion was markedly reduced by L-Glu at 10^?8 M, whereas L-Glu had little effect on the acid secretion stimulated by dimethyl-phenylpiperazinium (DMPP) at this concentration. However, at higher concentration, e.g., 10^?7 M, L-Glu also markedly reduced DMPP-induced acid secretion. Among L-Glu receptor agonists tested, quisqualic acid (QA) is most potent, followed by kainic acid (KA) and N-methyl-D-aspartic acid (NMDA) in inhibiting oxotremorine-stimulated acid secretion. Furthermore, this inhibitory effect of L-Glu on oxotremorine-stimulated acid secretion is blocked by 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), a specific non-NMDA receptor antagonist. All these results suggest that glutamatergic neurons are involved in the modulation of gastric acid secretion via ionotropic QA/KA receptors, probably through openings of Ca²⁺ channels. © 1994 Wiley-Liss, Inc.