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.     

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 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.     

Soluble guanylate cyclase and neuronal nitric oxide synthase colocalize in rat nucleus tractus solitarii

Nitric oxide has been implicated in transmission of cardiovascular signals in the nucleus tractus solitarii (NTS). Pharmacological studies suggest that activation of neurons by nitric oxide in the NTS may involve soluble guanylate cyclase (sGC). However, anatomical data supporting this suggestion have not been available. In this study, we tested the hypothesis that neurons and fibers containing neuronal nitric oxide synthase (nNOS) lie in close proximity to those containing sGC and the two enzymes colocalize in some neurons and fibers in the NTS. We perfused six rats and obtained brain stem sections for double immunofluorescent staining utilizing antibodies selective for sGC and for nNOS combined with confocal microscopy. The distribution and staining intensity of nNOS-immunoreactivity (IR) was similar to our earlier reports. IR of sGC was present in cell bodies, proximal dendrites and fibers of many brain stem regions. Strong sGC-IR was noted in the hypoglossal, dorsal motor nucleus of vagus and gracilis nuclei. The NTS exhibited moderate sGC-IR. Superimposed images showed that many NTS neurons contained both nNOS-IR and sGC-IR. The percentage of sGC-IR positive cells that were also nNOS-IR positive differed among NTS subnuclei. Similarly, the percentage of nNOS-IR positive cells that were also sGC positive differed among NTS subnuclei. Fibers stained for both nNOS-IR and sGC-IR were also present in NTS subnuclei. In addition, we identified fibers that were stained for nNOS-IR or sGC-IR alone and often found such singly labeled fibers apposed to each other. These data support our hypothesis and provide anatomical support for the suggestion that nitroxidergic activation of the NTS involves sGC.     

Coexistence of NMDA and AMPA receptor subunits with nNOS in the nucleus tractus solitarii of rat

We previously showed that most neuronal nitric oxide synthase (nNOS)-containing neurons in the nucleus tractus solitarii (NTS) contain NMDAR1, the fundamental subunit for functional N-methyl-D-aspartate (NMDA) receptors. Likewise, we found that almost all nNOS-containing neurons in the NTS contain GluR1, the calcium permeable AMPA receptor subunit. These data suggest that AMPA and NMDA receptors may colocalize in NTS neurons that contain nNOS. However, other investigators have suggested that non-NMDA receptors are located primarily on second-order neurons and NMDA receptors are located predominantly on higher-order neurons in NTS. We now seek to test the hypothesis that NMDA receptors, AMPA receptors and nNOS are colocalized in NTS cells. We performed triple fluorescent immunohistochemical staining of nNOS, NMDAR1 and GluR1, and performed confocal laser scanning microscopic analysis of the NTS. The distributions of nNOS immunoreactivity (IR), NMDAR1-IR and GluR1-IR in the NTS were similar to those we reported earlier. Superimposed images revealed that almost all NMDAR1-IR cells contained GluR1-IR and almost all GluR1-IR cells contained NMDAR1-IR. Some double-labeled cells were additionally labeled for nNOS-IR. All nNOS-IR neurons contained both GluR1-IR and NMDAR1-IR. These studies support our hypothesis that NMDA and AMPA receptors are colocalized in NTS neurons and are consistent with a role of both types of ionotropic receptors in transmission of afferent signals in NTS. In addition, these data provide support for an anatomical link between ionotropic glutamate receptors and nitric oxide in the NTS.     

Glutamatergic neurons say NO in the nucleus tractus solitarii

Both glutamate and nitric oxide (NO) may play an important role in cardiovascular reflex and respiratory signal transmission in the nucleus tractus solitarii (NTS). Pharmacological and physiological data have shown that glutamate and NO may be linked in mediating cardiovascular regulation by the NTS. Through tract tracing, multiple-label immunofluorescent staining, confocal microscopic, and electronic microscopic methods, we and other investigators have provided anatomical evidence that supports a role for glutamate and NO as well as an interaction between glutamate and NO in cardiovascular regulation in the NTS. This review article focuses on summarizing and discussing these anatomical findings. We utilized antibodies to markers of glutamatergic neurons and to neuronal NO synthase (nNOS), the enzyme that synthesizes NO in NTS neurons, to study the anatomical relationship between glutamate and NO in rats. Not only were glutamatergic markers and nNOS both found in similar subregions of the NTS and in vagal afferents, they were also frequently colocalized in the same neurons and fibers in the NTS. In addition, glutamatergic markers and nNOS were often present in fibers that were in close apposition to each other. Furthermore, N-methyl-d-aspartate (NMDA) type glutamate receptors and nNOS were often found on the same NTS neurons. Similarly, alpha-amino-3-hydroxy-5-methylisoxozole-proprionic acid (AMPA) type glutamate receptors also frequently colocalized with nNOS in NTS neurons. These findings support the suggestion that the interaction between glutamate and NO may be mediated both through NMDA and AMPA receptors. Finally, by applying tracer to the cut aortic depressor nerve (ADN) to identify nodose ganglion (NG) neurons that transmit cardiovascular signals to the NTS, we observed colocalization of vesicular glutamate transporters (VGluT) and nNOS in the ADN neurons. Thus, taken together, these neuroanatomical data support the hypothesis that glutamate and NO may interact with each other to regulate cardiovascular and likely other visceral functions through the NTS.     

Evidence for a glutamatergic input to pontine preganglionic neurons of the superior salivatory nucleus in rat

Parasympathetic preganglionic neurons of the superior salivatory nucleus (SSN), which projects to the pterygopalatine ganglion (PPG), modulate salivation, lacrimation, and cerebrovascular tone. Our previous studies suggest that excitatory projections from the nucleus tractus solitarii modulate cerebrovascular tone by actions on SSN neurons. In this study we sought to test the hypothesis that N-methyl-D-aspartate (NMDA) type glutamate receptors and vesicular glutamate transporters (VGLUT) are present in the SSN and that SSN neurons receive glutamatergic input. In six rats we injected tetramethylrhodamine dextran (TRD), a fluorescent tracer, unilaterally into the PPG to label SSN neurons. Four days later, rats were perfused and brain stem sections containing the SSN were processed for fluorescent immunohistochemistry for N-methyl-D-aspartate receptor subunit 1 (NMDAR1) and vesicular glutamate transporters (VGLUT1 and VGLUT2). Confocal laser scanning microscopy showed that 88+/-3% of TRD-labeled SSN neurons contained NMDAR1-immunoreactivity (IR). The surrounding neuropil contained numerous fibers labeled for VGLUT2-IR, but not VGLUT1-IR. Double fluorescent immunohistochemistry for NMDAR1 and VGLUT2 revealed that fibers containing VGLUT2-IR were often in close proximity to cell bodies or proximal dendrites of TRD-labeled SSN neurons that were positive for NMDAR1-IR. These studies support our hypothesis that NMDA receptors and VGLUT are present in the SSN. They further provide support for the suggestion that there are glutamatergic inputs to SSN neurons and would be consistent with an excitatory input that could regulate cerebrovascular tone.     

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.     

Anatomical evidence of non-parasympathetic cardiac nitrergic nerve fibres in rat

Neuronal nitric oxide synthase (nNOS)-derived nitric oxide (NO) plays a major role in the neural control of circulation and in many cardiovascular diseases. However, the exact mechanism of how NO regulates these processes is still not fully understood. This study was designed to determine the possible sources of nitrergic nerve fibres supplying the heart attempting to imply their role in the cardiac neural control. Sections of medulla oblongata, vagal nerve, its rootlets and nodose ganglia, vagal cardiac branches, Th₁-Th₅ spinal cord segments, dorsal root ganglia of C₈-Th₅ spinal nerves, and stellate ganglia from 28 Wistar rats were examined applying double immunohistochemical staining for nNOS combined with choline acetyltransferase (ChAT), peripherin, substance P, calcitonin gene-related peptide, tyrosine hydroxylase or myelin basic protein. Our findings show that the most abundant population of purely nNOS-immunoreactive (IR) neuronal somata (NS) was observed in the nodose ganglia (37.4 ± 1.3%). A high number of nitrergic NFs spread along the vagal nerve and entered its cardiac branches. All nitrergic neuronal somata (NS) in the nucleus ambiguus were simultaneously immunoreactive (IR) to ChAT and composed only a small subset of neurons (6%). In the dorsal nucleus of vagal nerve, biphenotypic nNOS-IR/ChAT-IR neurons composed 7.0 ± 1.0%, while small purely nNOS-IR neurons were scarce. Nitrergic NS were plentifully distributed within the nuclei of solitary tract. In the examined dorsal root and stellate ganglia, a few nitrergic NS were sporadically present. The majority of sympathetic NS in the intermediolateral nucleus were simultaneously immunoreactive for nNOS and ChAT. In conclusion, an abundant population of nitrergic NS in the nodose ganglion implies that neuronal NO is involved in afferent cardiac innervation. Nevertheless, nNOS-IR neurons identified within vagal nuclei may play a role in the transmission of preganglionic parasympathetic nerve impulses.     

Subcellular localization of neuronal nitric oxide synthase in the rat nucleus of the solitary tract in relation to vagal afferent inputs

In the nucleus of the solitary tract (NTS), nitric oxide (NO) modulates neuronal circuits controlling autonomic functions. A proposed source of this NO is via nitric oxide synthase (NOS) present in vagal afferent fibre terminals, which convey visceral afferent information to the NTS. Here, we first determined with electron microscopy that neuronal NOS (nNOS) is present in both presynaptic and postsynaptic structures in the NTS. To examine the relationship of nNOS to vagal afferent fibres the anterograde tracer biotinylated dextran amine was injected into the nodose ganglion and detected in brainstem sections using peroxidase-based methods. nNOS was subsequently visualised using a pre-embedding immunogold procedure. Ultrastructural examination revealed nNOS immunoreactivity in dendrites receiving vagal afferent input. However, although nNOS-immunoreactive terminals were frequently evident in the NTS, none were vagal afferent in origin. Dual immunofluorescence also confirmed lack of co-localisation. Nevertheless, nNOS immunoreactivity was observed in vagal afferent neurone cell bodies of the nodose ganglion. To determine if these labelled cells in the nodose ganglion were indeed vagal afferent neurones nodose ganglion sections were immunostained following application of cholera toxin B subunit to the heart. Whilst some cardiac-innervating neurones were also nNOS immunoreactive, nNOS was never detected in the central terminals of these neurones.These data show that nNOS is present in the NTS in both pre- and postsynaptic structures. However, these presynaptic structures are unlikely to be of vagal afferent origin. The lack of nNOS in vagal afferent terminals in the NTS, yet the presence in some vagal afferent cell bodies, suggests it is selectively targeted to specific regions of the same neurones.     

Nitrergic and VIPergic neurons in the choroid and ciliary ganglion of the duck Anis carina

Immunohistochemistry for neuronal nitric oxide synthase (nNOS) and vasoactive intestinal peptide (VIP), and NADPH diaphorase histochemistry, were applied to investigate neurons in the choroid and the ciliary ganglion of the muscovy duck Anis carina. Up to 1000 neurons in the choroid stained for NADPH diaphorase and showed virtually complete colocalization for nNOS immunoreactivity. Almost all of them co-stained for VIP, while about 90% of VIP immunoreactive cell bodies showed colocalization for nNOS. Two-thirds of the neurons were located, mostly singly, at nodes of a widemeshed nerve plexus in the suprachoroid and were only rarely grouped in ganglia of up to 3 neurons. Numerous varicose nNOS/NADPH-diaphorase-positive nerve fibers were seen around large arterial blood vessels. These fibers derived mainly from paravascular cell bodies that represented about one-third of all choroidal neurons and also displayed costaining for nitrergic markers and VIP. Colocalization of nNOS/NADPH-d and VIP could be demonstrated in most of the perivascular fibers, while slightly more VIP-positive axons in the suprachoroid plexus did not costain for nNOS/NADPH-d. Small-caliber blood vessels and those localized in the choriocapillaris were not endowed with VIP/nNOS/NADPH-diaphorase-positive fibers. A few reactive neuronal cell bodies were also found in ciliary nerves, while most ciliary axons were unstained. In the ciliary ganglion a small subpopulation of neurons showed VIP/nNOS/NADPH-diaphorase colocalization. There were also nNOS/ NADPH-d-positive cap-like terminals on ciliary ganglion cells. The presence of VIP/nNOS/NADPH-diaphorase positive neurons and nerve fibers in both the choroid and ciliary ganglion, and in the choroidal perivascular plexus, indicates peripheral nitrergic and VIPergic control of blood flow in the choroid of the duck.     

Expression of the vesicular glutamate transporters-1 and -2 in adult mouse dorsal root ganglia and spinal cord and their regulation by nerve injury

The expression of two vesicular glutamate transporters (VGLUTs), VGLUT1 and VGLUT2, was studied with immunohistochemistry in lumbar dorsal root ganglia (DRGs), the lumbar spinal cord and the skin of the adult mouse. About 12% and 65% of the total number of DRG neuron profiles (NPs) expressed VGLUT1 and VGLUT2, respectively. VGLUT1-immunoreactive (IR) NPs were usually medium- to large-sized, in contrast to a majority of small- or medium-sized VGLUT2-IR NPs. Most VGLUT1-IR NPs did not coexpress calcitonin gene-related peptide (CGRP) or bound isolectin B4 (IB4). In contrast, approximately 31% and approximately 42% of the VGLUT2-IR DRG NPs were also CGRP-IR or bound IB4, respectively. Conversely, virtually all CGRP-IR and IB4-binding NPs coexpressed VGLUT2. Moderate colocalization between VGLUT1 and VGLUT2 was also observed. Sciatic nerve transection induced a decrease in the overall number of VGLUT1- and VGLUT2-IR NPs (both ipsi- and contralaterally) and, in addition, a parallel, unilateral increase of VGLUT2-like immunoreactivity (LI) in a subpopulation of mostly small NPs. In the dorsal horn of the spinal cord, strong VGLUT1-LI was detected, particularly in deep dorsal horn layers and in the ventral horns. VGLUT2-LI was abundant throughout the gray spinal matter, 'radiating' into/from the white matter. A unilateral dorsal rhizotomy reduced VGLUT1-LI, while apparently leaving unaffected the VGLUT2-LI. Transport through axons for both VGLUTs was confirmed by their accumulation after compression of the sciatic nerve or dorsal roots. In the hind paw skin, abundant VGLUT2-IR nerve fibers were observed, sometimes associated with Merkel cells. Lower numbers of VGLUT1-IR fibers were also detected in the skin. Some VGLUT1-IR and VGLUT2-IR fibers were associated with hair follicles. Based on these data and those by Morris et al. [Morris JL, Konig P, Shimizu T, Jobling P, Gibbins IL (2005) Most peptide-containing sensory neurons lack proteins for exocytotic release and vesicular transport of glutamate. J Comp Neurol 483:1-16], we speculate that virtually all DRG neurons in adult mouse express VGLUTs and use glutamate as transmitter.     

Upregulation of NMDA receptor and neuronal NADPH-d/NOS expression in the nodose ganglion of acute hypoxic rats

Nitric oxide may serve as a neuronal messenger in the regulation of cardiorespiratory function via the N-methyl-D-aspartate (NMDA) receptor-mediated neuronal nitric oxide synthase (nNOS) activation. Since hypoxic stress would drastically influence the cardiorespiratory function, the present study aimed to examine if the expression of nNOS and NMDA receptor subunit 1 (NMDAR1) in the nodose ganglion (NG) would alter under different extents of hypoxia treatment. The nicotinamine adenine dinucleotide phosphate-diaphorase (NADPH-d) histochemistry, nNOS and NMDAR1 immunofluorescence were used to examine nNOS and NMDAR1 expression in the NG following exposing of adult rats in the altitude chamber (0.27 atm, PO(2)=43 torr) for 2 and 4 h. The present results showed that NADPH-d, nNOS and NMDAR1 reactivities were co-localized in the NG under normoxic and hypoxic environment. Quantitative evaluation revealed that about 43% of neurons in the NG showed positive response for NADPH-d/nNOS and NMDAR1 reactivities. However, in animals subjected to hypoxia, both the percentage and the staining intensity of NADPH-d/nNOS and NMDAR1 labeled neurons were drastically increased. The percentage of NADPH-d/nNOS and NMDAR1-immunoreactive neurons in the NG was raised to 68% as well as 77%, respectively, following 2 and 4 h of hypoxic exposure. The magnitude of up-regulation was positively correlated with the duration of hypoxic periods. No significant cell loss was observed under this experimental paradigm. These findings suggest that different extents of hypoxia might induce the higher expression of nNOS and NMDAR1 in the NG, which could contribute to the neuronal integration as responding to the different physiological demands under hypoxic stress.     

No colocalization of immunoreactivities for VIP and neuronal NOS, and a differential relation to cGMP-immunoreactivity in bovine penile smooth muscle

The distribution of immunoreactivity (IR) for the neuropeptide vasoactive intestinal polypeptide (VIP) and neuronal nitric oxide synthase (nNOS) in the bovine retractor penis muscle (RP) and penile artery (PA) was studied by using two different methods. The distribution of these immunoreactivities was also compared with that of the immunoreactivity for cyclic guanosine monophosphate (cGMP). In both tissues the nerve fibers and terminals immunoreactive for VIP had a distribution that was completely different from that of the nerve fibers and terminals immunoreactive for nNOS. This contrasts with the previous observations in penile smooth muscle of other species. In the RP, as well as in the PA, many of the VIP-IR fibers were also immunoreactive for neurofilaments (NF), whereas the nNOS-IR fibers were consistently devoid of NF-IR. Stimulation with sodium nitroprusside, a nitric oxide donor, considerably increased cGMP-IR in the smooth muscle cells in both RP and PA, and in several nerve fibers in PA. Many of these cGMP-IR nerve fibers exhibited nNOS-IR, whereas none of them was immunoreactive for VIP. Our results suggest that the degree of coexistence of VIP-IR and nNOS-IR in the nerve fibers and terminals innervating penile smooth muscle show wide species differences. They also suggest that the mechanisms by which VIP could be involved in neurogenic penile erection may vary between species.     

Collateral damage and compensatory changes after injection of a toxin targeting neurons with the neurokinin-1 receptor in the nucleus tractus solitarii of rat

Injection into the nucleus tractus solitarii (NTS) of toxins that target substance P (SP) receptors ablates neurons that express neurokinin-1 (NK1) receptors, attenuates baroreflexes, and results in increased lability of arterial pressure. We and others have shown that the toxin leads to loss of neurons containing SP receptors and loss of GABAergic neurons in the NTS; but given that neither type neuron is thought to be integral to baroreflex transmission in NTS, mechanisms responsible for the cardiovascular changes remained unclear. Because NK1 receptors colocalize with N-methyl-d-aspartate (NMDA) receptors and alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors in NTS and because glutamate transmission may be integral to baroreflex transmission in the NTS we hypothesized that the toxic lesions may interrupt mechanisms for glutamate transmission. Interruption of those mechanisms could be responsible for the cardiovascular effects. We tested the hypothesis by performing fluorescent immunohistochemistry, confocal microscopy and image analysis after injecting stabilized SP-SAP (SSP-SAP) unilaterally into the NTS. We assessed changes in immunoreactivity (IR) of NMDA receptor subunit 1 (NMDAR1), AMPA receptor subunit 2 (GluR2), and 3 types of vesicular glutamate transporters (VGluT) as well as IR of gamma-aminobutyric acid receptors type b (GABAb), neuronal nitric oxide synthase (nNOS), tyrosine hydroxylase (TH), and protein gene product 9.5 (PGP 9.5), a neuronal marker, in the NTS. When compared to that of the same section of the un-injected NTS, IR decreased significantly in the injected side for NMDAR1 (p<0.01), GluR2 (p<0.01), VGluT3 (p<0.01), GABAb (p<0.001), and PGP9.5 (p<0.001). In contrast, IR for VGluT1 (p<0.001), VGluT2 (p<0.001), nNOS (p<0.001), and TH (p<0.001) increased significantly. We conclude that pathologic effects following ablation of neurons with NK1 receptors in NTS may result from interruption of neurotransmission through other neurochemical systems associated with NK1 receptors-containing neurons.     

Differential expression of vesicular glutamate transporters by vagal afferent terminals in rat nucleus of the solitary tract: projections from the heart preferentially express vesicular glutamate transporter 1

The central projections and neurochemistry of vagal afferent neurones supplying the heart in the rat were investigated by injecting cholera toxin B-subunit into the pericardium. Transganglionically transported cholera toxin B-subunit was visualized in the medulla oblongata in axons and varicosities that were predominantly aggregated in the dorsomedial, dorsolateral, ventrolateral and commissural subnuclei of the caudal nucleus of the solitary tract. Unilateral vagal section in control rats prevented cholera toxin B-subunit labeling on the ipsilateral side of the nucleus of the solitary tract. Fluorescent and electron microscopic dual labeling showed colocalization of immunoreactivity for vesicular glutamate transporter 1, but only rarely vesicular glutamate transporters 2 or 3 with cholera toxin B-subunit in terminals in nucleus of the solitary tract, suggesting that cardiac vagal axons release glutamate as a neurotransmitter. In contrast, populations of vagal afferent fibers labeled by injection of cholera toxin B-subunit, tetra-methylrhodamine dextran or biotin dextran amine into the aortic nerve, stomach or nodose ganglion colocalized vesicular glutamate transporter 2 more frequently than vesicular glutamate transporter 1. The presence of other neurochemical markers of primary afferent neurones was examined in nucleus of the solitary tract axons and nodose ganglion cells labeled by pericardial cholera toxin B-subunit injections. Immunoreactivity for a 200-kDa neurofilament protein in many large, cholera toxin B-subunit-labeled nodose ganglion cells indicated that the cardiac afferent fibers labeled are mostly myelinated, whereas binding of Griffonia simplicifolia isolectin B4 to fewer small cholera toxin B-subunit-labeled ganglion cells suggested that tracer was also taken up by some non-myelinated axons. A few labeled nucleus of the solitary tract axons and ganglion cells were positive for substance P and calcitonin gene-related peptide, which are considered as peptide markers of nociceptive afferent neurones. These data suggest that the population of cardiac vagal afferents labeled by pericardial cholera toxin B-subunit injection is neurochemically varied, which may be related to a functional heterogeneity of baroreceptive, chemoreceptive and nociceptive afferent fibers. A high proportion of cardiac neurones appear to be glutamatergic, but differ from other vagal afferents in expressing vesicular glutamate transporter 1.     

Topographical distributions of endomorphinergic pathways from nucleus tractus solitarii to periaqueductal gray in the rat

In the central nervous system (CNS), endomorphin 1 (EM1)- and endomorphin 2 (EM2)-containing neuronal cell bodies have been found in the nucleus tractus sollitarii (NTS) and the hypothalamus, and EMergic fibers and terminals are distributed widely in many regions of the CNS, including the periaqueductal gray (PAG). The aim of the present study was to examine whether EM-expressing neurons in the NTS of the rat send their axons to the PAG, and determine whether the EMergic pathway from the NTS to the PAG is topographic by using. Immunofluorescent staining for EM1 or EM2 combined with retrograde and anterograde tract-tracing methods. The results showed that after injecting tetramethyl rhodamine dextran-amine (TMR) into the ventrolateral or lateral column of the PAG, some EM1- or EM2-immunoreactive (IR) neurons in the NTS were retrogradely labeled with TMR, and the majority of the EM-IR/TMR double-labeled neurons were mainly distributed in the medial and commissural subnuclei of the NTS. Following injection of biotinylated dextran amine (BDA) into the medial or commissural subnucleus of the NTS, EM1-IR/BDA and EM2-IR/BDA double-labeled fibers and terminals were mainly distributed in the ventrolateral or lateral column of the PAG, respectively. The results indicate that EMergic pathway from the NTS to PAG is topographically organized, and suggest that EMs released from NTS to PAG projecting terminals may bind to μ-opioid receptor on the PAG neurons, and thereby contribute to various functions.     

Distribution of calbindin-D28k, neuronal nitric oxide synthase, and nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-d) in the lateral nucleus of the sheep amygdaloid complex

This study describes calbindin-D28k (CB), neuronal nitric oxide synthase (nNOS), and nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-d) expression in the lateral nucleus of the sheep amygdaloid complex. Double immunofluorescence protocol was used in order to determine whether there is colocalization of CB and nNOS. The CB-immunoreactive (IR) neuronal population was composed especially of non-pyramidal neurons, but a few pyramidal cells were also present. The non-pyramidal neurons showed a multipolar and, occasionally, a fusiform morphology. The comparison between single-labeled CB-IR non-pyramidal neurons and cells belonging to CB-IR neuronal population showed they were identical for morphology, mean size, and distribution. The single-labeled CB-IR non-pyramidal neurons were only the 17.8% of the total non-pyramidal neurons counted. The nNOS-IR neuronal population was represented by non-pyramidal multipolar and fusiform neurons. Single-labeled nNOS-IR non-pyramidal neurons had the same morphology, mean area, and distribution as cells belonging to nNOS-IR neuronal population. Single-labeled nNOS-IR non-pyramidal neurons were more numerous than single-labeled CB-IR, and represented the 73.7% of total non-pyramidal neurons counted. NADPH-d-positive cells had the same morphology and distribution as the nNOS-IR neurons. Double immunolabeling (CB/nNOS) was found mostly in non-pyramidal multipolar neurons and only in a few non-pyramidal fusiform cells. These neurons had a mean perikaryal area significantly higher and significantly smaller than that of single-labeled nNOS and single-labeled CB-IR non-pyramidal neurons, respectively. CB and nNOS coexist only in a minority of non-pyramidal neurons (8.5%). The 32.4% of all CB-IR non-pyramidal neurons were nNOS-positive; only 10.4% of nNOS-IR non-pyramidal neurons were CB-positive. These results indicate that CB and nNOS are expressed by selective neurons and that the majority of nNOS-IR non-pyramidal neurons are lacking in CB.     

Acid-sensing ion channel 1 and nitric oxide synthase are in adjacent layers in the wall of rat and human cerebral arteries

Extracellular acidification activates a family of proteins known as acid-sensing ion channels (ASICs). One ASIC subtype, ASIC type 1 (ASIC1), may play an important role in synaptic plasticity, memory, fear conditioning and ischemic brain injury. ASIC1 is found primarily in neurons, but one report showed its expression in isolated mouse cerebrovascular cells. In this study, we sought to determine if ASIC1 is present in intact rat and human major cerebral arteries. A potential physiological significance of such a finding is suggested by studies showing that nitric oxide (NO), which acts as a powerful vasodilator, may modulate proton-gated currents in cultured cells expressing ASIC1s. Because both constitutive NO synthesizing enzymes, neuronal nitric oxide synthase (nNOS) and endothelial NOS (eNOS), are expressed in cerebral arteries we also studied the anatomical relationship between ASIC1 and nNOS or eNOS in both rat and human cerebral arteries. Western blot analysis demonstrated ASIC1 in cerebral arteries from both species. Immunofluorescent histochemistry and confocal microscopy also showed that ASIC1-immunoreactivity (IR), colocalized with the smooth muscle marker alpha-smooth muscle actin (SMA), was present in the anterior cerebral artery (ACA), middle cerebral artery (MCA), posterior cerebral artery (PCA) and basilar artery (BA) of rat and human. Expression of ASIC1 in cerebral arteries is consistent with a role for ASIC1 in modulating cerebrovascular tone both in rat and human. Potential interactions between smooth muscle ASIC1 and nNOS or eNOS were supported by the presence of nNOS-IR in the neighboring adventitial layer and the presence of nNOS-IR and eNOS-IR in the adjacent endothelial layer of the cerebral arteries.     

Expression of Group I metabotropic glutamate receptors on phenotypically different cells within the nucleus of the solitary tract in the rat

Group I metabotropic glutamate receptors (mGluRs) are G-coupled receptors that modulate synaptic activity. Previous studies have shown that Group I mGluRs are present in the nucleus of the solitary tract (NTS), in which many visceral afferents terminate. Microinjection of selective Group I mGluR agonists into the NTS results in a depressor response and decrease in sympathetic nerve activity. There is, however, little evidence detailing which phenotypes of neurons within the NTS express Group I mGluRs. In brainstem slices, we performed immunohistochemical localization of Group I mGluRs and either glutamic acid decarboxylase 67 kDa isoform (GAD67), neuronal nitric oxide synthase (nNOS) or tyrosine hydroxylase (TH). Fluoro-Gold (FG, 2%; 15 nl) was microinjected in the caudal ventrolateral medulla (CVLM) of the rat to retrogradely label NTS neurons that project to CVLM. Group I mGluRs were distributed throughout the rostral–caudal extent of the NTS and were found within most NTS subregions. The relative percentages of Group I mGluR expressing neurons colabeled with the different markers were FG (6.9±0.7) nNOS (5.6±0.9), TH (3.9±1.0), and GAD67 (3.1±1.4). The percentage of FG containing cells colabeled with Group I mGluR (13.6±2.0) was greater than the percent colabeled with GAD67 (3.1±0.5), nNOS (4.7±0.5), and TH (0.1±0.08). Cells triple labeled for FG, nNOS, and Group I mGluRs were identified in the NTS. Thus, these data provide an anatomical substrate by which Group I mGluRs could modulate activity of CVLM projecting neurons in the NTS.