Li-Hsien Lin

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.

Identification and localization of cell types that express endothelial and neuronal nitric oxide synthase in the rat nucleus tractus solitarii

Numerous studies have suggested that nitric oxide (NO) in the nucleus tractus solitarii (NTS) participates in modulating cardiovascular function. Nitric oxide synthase (NOS), the enzyme responsible for synthesis of NO, exists in 3 isoforms: endothelial NOS (eNOS), neuronal NOS (nNOS), and inducible NOS (iNOS). Although the distribution of nNOS in the NTS has been well documented, the distribution of eNOS in the NTS has not. Because recent studies have shown that eNOS may contribute to regulation of baroreceptor reflexes and arterial pressure, we examined the distribution of eNOS and the types of cells that express it in rat NTS by using multiple labels for immunofluorescent staining and confocal microscopy. Immunoreactivity (IR) for eNOS and nNOS was found in cells and processes in all NTS subnuclei, but eNOS-IR was more uniformly distributed than was nNOS-IR. Although structures containing either eNOS-IR or nNOS-IR were often present in close proximity, they never contained both isoforms. Almost all eNOS-IR positive structures, but no nNOS-IR positive structures, contained IR for the glial marker glial fibrillary acidic protein. Furthermore, while all nNOS-IR positive cells contained IR for the neuronal marker neuronal nuclear antigen (NeuN), none of the eNOS-IR positive cells contained NeuN-IR. We conclude that eNOS in the NTS is present only in astrocytes and endothelial cells, not in neurons. Our data complement previous physiological studies and suggest that although NO from nNOS may modulate neurotransmission directly in the NTS, NO from eNOS in the NTS may modulate cardiovascular function through an interaction between astrocytes and neurons.

Apposition of neuronal elements containing nitric oxide synthase and glutamate in the nucleus tractus solitarii of rat: a confocal microscopic analysis

The distribution of glutamate and neuronal nitric oxide synthase in the rat nucleus tractus solitarii was investigated by double fluorescent immunohistochemistry combined with confocal laser scanning microscopy. Cells and fibers that exhibited neuronal nitric oxide synthase immunoreactivity alone, glutamate immunoreactivity alone or both immunolabels were present in all subnuclei of the nucleus tractus solitarii, but staining intensities differed between the subnuclei. The percentages of double-labeled glutamate-immunoreactive cells also differed between the subnuclei. The central subnucleus contained the highest percentage of double-labeled glutamate-immunoreactive cells and the medial subnucleus contained the lowest. The percentages of double-labeled neuronal nitric oxide synthase-immunoreactive neurons likewise differed between the subnuclei. The central subnucleus contained the highest percentage of double-labeled neuronal nitric oxide synthase-immunoreactive neurons and the commissural subnucleus contained the lowest. Because of our interest in cardiovascular regulation, the anatomical relationship between glutamate-immunoreactive and neuronal nitric oxide synthase-immunoreactive fibers in the dorsolateral and commissural subnuclei was further examined at higher magnification. Close appositions were observed between neuronal nitric oxide synthase-immunoreactive and glutamate-immunoreactive fibers, between double-labeled and glutamate-immunoreactive fibers, and between neuronal nitric oxide synthase-immunoreactive and double-labeled fibers. We recognized that a single visual perspective might cause labeled fibers that pass in close proximity to appear to make contact. Therefore, we constructed three-dimensional images from serial optical sections obtained from the dorsolateral and commissural subnuclei by means of a confocal scanning microscope. Rotation of the three-dimensional images caused some fibers that had seemed to be in close apposition to other structures to separate from those structures. In contrast, some glutamate-immunoreactive and some neuronal nitric oxide synthase-immunoreactive fibers remained in close apposition regardless of the angle at which they were viewed. This study supports there being an anatomical link between glutamatergic and nitroxidergic systems in the nucleus tractus solitarii. Recognized physiological interactions between the two systems could occur through such a link.

N-methyl-d-aspartate receptors on neurons that synthesize nitric oxide in rat nucleus tractus solitarii

The aim of this study was to determine whether neuronal nitric oxide synthase and N-methyl-D-aspartate receptors are co-localized in the rat nucleus tractus solitarii. Such co-localization would support the hypothesis that nitric oxide participates in nucleus tractus solitarii-mediated functions, such as cardiovascular regulation, by a link to N-methyl-D-aspartate receptors. We used double fluorescent immunohistochemistry using antibodies against neuronal nitric oxide synthase and N-methyl-D-aspartate receptor subunit 1, the fundamental subunit for functional N-methyl-D-aspartate receptors. Labeled brainstem sections were examined with confocal laser scanning microscopy. Most of the N-methyl-D-aspartate receptor subunit 1 immunoreactivity was in cell bodies and proximal dendrites of the numerous labeled cells in the brainstem. High levels of N-methyl-D-aspartate receptor subunit 1 immunoreactivity were present in the dorsal motor nucleus of vagus, hypoglossal nucleus and nucleus ambiguus. All subnuclei of the nucleus tractus solitarii contained moderate levels of N-methyl-D-aspartate receptor subunit 1 immunoreactivity. The distribution of neuronal nitric oxide synthase immunoreactivity in the nucleus tractus solitarii was similar to that described in earlier reports. Superimposition of images revealed that almost all neuronal nitric oxide synthase immunoreactive neurons in the nucleus tractus solitarii contained N-methyl-D-aspartate receptor subunit 1 immunoreactivity, but a lesser portion of N-methyl-D-aspartate receptor subunit 1-immunoreactive cells contained neuronal nitric oxide synthase immunoreactivity. Although all nucleus tractus solitarii subnuclei contained double-labeled neurons, the central subnucleus exhibited the highest density of double-labeled neurons.Co-localization of neuronal nitric oxide synthase and N-methyl-D-aspartate receptor subunit 1 in the nucleus tractus solitarii provides anatomical support for the hypothesis that N-methyl-D-aspartate receptor activation can affect nucleus tractus solitarii-controlled functions via actions on neurons that synthesize nitric oxide.

Nitroxidergic Influences on Cardiovascular Control by NTS: A Link with Glutamate

Abstract: Glutamate (GLU) receptor activation, which is important in cardiovascular reflex transmission through the nucleus tractus solitarii (NTS), leads to release of nitric oxide (NO·) from central nitroxidergic neurons. Therefore, we hypothesized that GLU and NO· are linked in cardiovascular control by NTS. We first sought to determine if NO· released into NTS led to cardiovascular changes like those produced by GLU and found that the nitrosothiol S‐nitrosocysteine, but not NO· itself or other NO· donors, elicited such responses in anesthetized rats. The responses were dependent on activation of soluble guanylate cyclase but, not being affected by a scavenger of NO·, likely did not depend on release of NO· into the extracellular space. Responses to ionotropic GLU agonists in NTS, like those to S‐nitrosocysteine, were inhibited by inhibition of soluble guanylate cyclase. Inhibition of neuronal NO· synthase (nNOS) also inhibited responses to ionotropic GLU agonists. The apparent physiologic link between GLU and NO· mechanisms in NTS was further supported by anatomical studies that demonstrated frequent association between GLU‐containing nerve terminals and neurons containing nNOS. Furthermore, GLU receptors were often found on NTS neurons that were immunoreactive for nNOS. The anatomical relationships between GLU and nNOS and GLU receptors and nNOS were more pronounced in some subnuclei of NTS than in others. While seen in subnuclei that are known to receive cardiovascular afferents, the association was even more prominent in subnuclei that receive gastrointestinal afferents. These studies support a role for nitroxidergic neurons in mediating cardiovascular and other visceral reflex responses that result from release of GLU into the NTS.

Localization of vesicular glutamate transporters and neuronal nitric oxide synthase in rat nucleus tractus solitarii.

Previously we reported that glutamate and neuronal nitric oxide synthase (nNOS) colocalize in neurons of the nucleus tractus solitarii (NTS). That finding provided anatomical support for the suggestion that nitric oxide and glutamate interact in cardiovascular regulation by the NTS. Here we test the hypothesis that nNOS colocalizes with vesicular glutamate transporters (VGluT1 and VGluT2) in the NTS. Immunoreactivity (IR) for VGluT better identifies glutamatergic terminals than does glutamate-IR, which may label metabolic as well as transmitter stores of the amino acid. We used fluorescent immunohistochemistry combined with confocal laser scanning microscopy to study IR for VGluT1, VGluT2 and nNOS in rat NTS. A high density of VGluT1-IR positive fibers was present in the gracilis and cuneatus nuclei while in the NTS we found a moderate density in the lateral and interstitial subnuclei and a low density in the dorsolateral, ventral and intermediate subnuclei. The medial, central, commissural and gelatinosus subnuclei contained few VGluT1-IR containing fibers. Thus, VGluT1 containing fibers are not prominent in portions of the NTS where cardiovascular afferent fibers terminate. In contrast, we found a high density of VGluT2-IR containing fibers in the gelatinosus subnucleus and subpostremal area and a moderate density in cardiovascular regions such as the dorsolateral and medial subnuclei as well as in the central and lateral subnuclei. We found a low density in the ventral, intermediate, interstitial and commissural subnuclei. VGluT1-IR and VGluT2-IR rarely colocalized in fibers within the NTS. VGluT1-IR did not colocalize with nNOS, but VGluT2-IR and nNOS-IR colocalized in fibers in all NTS subnuclei. When compared with the other NTS subnuclei, the dorsolateral, gelatinosus and subpostremal subnuclei had higher frequencies of colocalization of VGluT2-IR and nNOS-IR. VGluT2-IR positive fibers were also apposed to nNOS-IR positive fibers throughout the NTS. These data support our hypothesis and confirm that glutamatergic fibers in the NTS contain nNOS.

Up-regulation of nitric oxide synthase and its mRNA in vagal motor nuclei following axotomy in rat

Effects of vagotomy on nitric oxide synthase (NOS) protein and mRNA levels in the dorsal motor nucleus of vagus (DMV) and nucleus ambiguus (NA) of rats were examined by nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) staining, brain NOS (bNOS) immunostaining and in situ hybridization. NADPH-d staining and bNOS immunoreactivity increased in neurons of the ipsilateral DMV and NA 5, 10, and 20 days after vagotomy. These changes were not observed in unoperated or sham-operated rats. In situ hybridization showed that bNOS mRNA levels were also elevated in neurons of DMV and NA on the operated side. Our results suggest that transection of vagal efferents up-regulates bNOS and its mRNA expression in the DMV and NA.

Colocalization of neurokinin-1, N-methyl-d-aspartate, and AMPA receptors on neurons of the rat nucleus tractus solitarii

Substance P (SP) and glutamate are implicated in cardiovascular regulation by the nucleus tractus solitarii (NTS). Our earlier studies suggest that SP, which acts at neurokinin 1 (NK1) receptors, is not a baroreflex transmitter while glutamate is. On the other hand, our recent studies showed that loss of NTS neurons expressing NK1 receptors leads to loss of baroreflex responses and increased blood pressure lability. Furthermore, studies have suggested that SP may interact with glutamate in the NTS. In this study, we sought to test the hypothesis that NK1 receptors colocalize with glutamate receptors, either N-methyl-d-aspartate (NMDA) receptors or AMPA receptors or both in the NTS. We performed double-label immunofluorescent staining for NK1 receptors and either N-methyl-d-aspartate receptor subunit 1 (NMDAR1) or AMPA specific glutamate receptor subunit 2 (GluR2) in the rat NTS. Because vesicular glutamate transporter 2 (VGLUT2) containing fibers are prominent in portions of the NTS where cardiovascular afferent fibers terminate, we also performed double-label immunofluorescent staining for NK1 receptors and VGLUT2. Confocal microscopic images showed that NK1 receptors-immunoreactivity (IR) and NMDAR1-IR colocalized in the same neurons in many NTS subnuclei. Almost all NTS neurons positive for NK1 receptor-IR also contained NMDAR1-IR, but only 53.4% to 74.8% of NMDAR1-IR positive neurons contained NK1 receptors-IR. NK1 receptor-IR and GluR2-IR also colocalized in many neurons in NTS subnuclei. A majority of NK1 receptor-IR positive NTS neurons also contained GluR2-IR, but only 45.8% to 73.9% of GluR2-IR positive NTS neurons contained NK1 receptors-IR. Our results also showed that fibers labeled for VGLUT2-IR were in close apposition to fibers and neurons labeled for NK1 receptor-IR. The data support our hypothesis, provide an anatomical framework for glutamate and SP interactions, and may explain the loss of baroreflexes when NTS neurons, which could respond to glutamate as well as SP, are killed.

Vesicular glutamate transporters and neuronal nitric oxide synthase colocalize in aortic depressor afferent neurons

The aortic depressor nerve (ADN) primarily transmits baroreceptor signals from the aortic arch to the nucleus tractus solitarii. Cell bodies of neurons that send peripheral fibers to form the ADN are located in the nodose ganglion (NG). Studies have implicated glutamate and nitric oxide in transmission of baroreflex signals; therefore, we tested the hypothesis that ADN neurons contain either vesicular glutamate transporters (VGLUTs) or neuronal nitric oxide synthase (nNOS) or both. We applied a fluorescent tracer, tetramethyl rhodamine dextran (TRD), to rat ADN to identify ADN neurons and then performed immunofluorescent labeling for nNOS and VGLUTs 1, 2, and 3 in NG sections. We found that VGLUT2-immunoreactivity (IR) and VGLUT3-IR was present in a significantly higher proportion of TRD positive neurons than in TRD negative neurons. In contrast, the percentage of TRD positive neurons containing VGLUT1-IR or nNOS-IR did not differ from that of TRD negative neurons. We also observed that the percentage of TRD positive neurons containing both VGLUT2-IR and nNOS-IR and the percentage of TRD positive neurons containing both VGLUT3-IR and nNOS-IR were significantly higher than that of TRD negative neurons. On the other hand, colocalization of VGLUT1-IR and nNOS-IR in TRD positive neurons did not differ from that of TRD negative neurons. These results support our hypothesis and suggest prominent roles of VGLUT2-IR containing neurons and VGLUT3-IR containing neurons in transmitting cardiovascular signals via the ADN to the brain stem.

Colocalization of GluR1 and neuronal nitric oxide synthase in rat nucleus tractus solitarii neurons.

Previously we demonstrated that glutamate and neuronal nitric oxide synthase (nNOS) containing neuronal elements are frequently apposed in subnuclei of the rat nucleus tractus solitarii. It is known that glutamate receptors (GluRs) of the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) subtype participate in cardiovascular regulation by the nucleus tractus solitarii and that responses to AMPA receptor activation may be linked to NO. Therefore, in the present study, we further tested the hypothesis that the calcium-permeable subunit GluR1 of AMPA type GluRs and nNOS are colocalized in neurons of the nucleus tractus solitarii. Distribution of GluR1 and nNOS in rat nucleus tractus solitarii was investigated by double fluorescent immunohistochemistry combined with confocal laser scanning microscopy. Numerous GluR1 immunoreactive cells and fibers were present in subnuclei of the nucleus tractus solitarii. The staining intensity of GluR1 immunoreactive cells varied among subnuclei. Cells in the interstitial subnucleus contained the highest GluR1 staining intensity. A moderate intensity of staining was present in the intermediate, dorsolateral, ventral, and commissural subnuclei. A slightly lower level of GluR1 immunoreactivity was present in cells of the medial subnucleus. Cells in the central subnucleus contained a low level of GluR1 immunoreactivity. The staining intensity of GluR1 immunoreactive fibers also varied among subnuclei. Distribution of nNOS immunoreactivity in the nucleus tractus solitarii and other brain stem areas was the same as in our earlier reports. Superimposition of confocal images of nNOS immunoreactivity and GluR1 immunoreactivity allowed us to identify double-labeled structures. Nearly all neurons that were immunoreactive for nNOS contained GluR1 immunoreactivity, but only a proportion of GluR1 immunoreactive cells contained nNOS immunoreactivity. Double-labeled neurons were present in all subnuclei of the nucleus tractus solitarii. The percentages of GluR1 immunoreactive cells that also contained nNOS immunoreactivity differed among subnuclei of the nucleus tractus solitarii. Fibers that labeled for nNOS alone, GluR1 alone or both were present among labeled cells in these subnuclei. These data support the hypothesis that GluR1 and nNOS are colocalized in neurons of nucleus tractus solitarii. The demonstration of this anatomical relationship provides further anatomical support for the hypothesis that activation of AMPA receptors on neurons that synthesize NO in the nucleus tractus solitarii contributes to autonomic regulation.