Feline immunodeficiency virus as a gene transfer vector in the rat nucleus tractus solitarii.

Gene transfer has been used to examine the role of putative neurotransmitters in the nucleus tractus solitarii (NTS). Most such studies used adenovirus vector-mediated gene transfer although adenovirus vector transfects both neuronal and non-neuronal cells. Successful transfection in the NTS has also been reported with lentivirus as the vector. Feline immunodeficiency virus (FIV), a lentivirus, may preferentially transfect neurons and could be a powerful tool to delineate physiological effects produced by altered synthesis of transmitters in neurons. However, it has not been studied in NTS. Therefore, we sought to determine whether FIV transfects rat NTS cells and to define the type of cell transfected. We found that injection of FIV encoding LacZ gene (FIVLacZ) into the NTS led to transfection of numerous NTS cells. Injection of FIVLacZ did not alter immunoreactivity (IR) for neuronal nitric oxide synthase, which we have shown resides in NTS neurons. A majority (91.7 +/- 3.9%) of transfected cells contained IR for neuronal nuclear antigen, a neuronal marker; 2.1 +/- 3.8% of transfected cells contained IR for glial fibrillary acidic protein, a glial marker. No transfected neurons or fibers were observed in the nodose ganglion, which sends afferents to the NTS. We conclude that FIV almost exclusively transfects neurons in the rat NTS from which it is not retrogradely transported. The cell-type specificity of FIV in the NTS may provide a molecular method to study local physiological functions mediated by potential neurotransmitters in the NTS.

Evidence for L-glutamate as the neurotransmitter of baroreceptor afferent nerve fibers.

Microinjection of L-glutamate into the intermediate nucleus tractus solitarii in anesthetized rats elicits hypotension, bradycardia, and apnea, simulating baroreceptor reflexes. Ablation of the nodose ganglion results in selective reduction of high-affinity uptake of L-glutanate in the nucleus tractus solitarii. L-Glutamate may be the neurotransmitter of afferent nerve fibers from arterial baroreceptors.

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.

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.

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.

Nitroxidergic neurons in rat nucleus tractus solitarii express vesicular glutamate transporter 3.

Earlier we reported that glutamate transporter (VGLUT) 2 and neuronal nitric oxide synthase (nNOS) are colocalized in some fibers and are present in apposing fibers in the nucleus tractus solitarii (NTS). Those findings provided anatomical support for a hypothesized physiological link between glutamate and nitric oxide (NO.) in the NTS. Recently a third class of VGLUT, VGLUT3, was identified, but its distribution in NTS and its anatomical relationship with nNOS have not been shown. In this study we tested the hypothesis that neurons and fibers containing VGLUT3 lie in close proximity to those containing nNOS and that both proteins colocalize in some neurons and fibers in the NTS. We perfused rats and obtained brain stem sections and nodose ganglion sections for immunofluorescent staining analyzed by confocal microscopy. The NTS contained moderate VGLUT3-immunoreactivity (IR), with the intermediate, medial and interstitial subnuclei containing higher VGLUT3-IR than other subnuclei. Although all three forms of VGLUT were present in the NTS, VGLUT3-IR was not colocalized with either VGLUT1-IR or VGLUT2-IR in either processes or cells in the brain stem. Cells and processes containing both VGLUT3-IR and nNOS-IR were noted in all NTS subnuclei and in the nodose ganglion. Triple immunofluorescent staining revealed that cells double-labeled for nNOS-IR and VGLUT3-IR were all additionally labeled for neuronal nuclear antigen (NeuN), a neuronal marker. These findings support our hypothesis that neurons and fibers containing VGLUT3 lie in close proximity to those containing nNOS and that both proteins colocalize in some neurons and fibers in the NTS.

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.

Glycine microinjected in the rat dorsal vagal nucleus increases arterial pressure.

Microinjections (25 nl) of glycine into the dorsal motor nucleus of the vagus in 21 rats elicited dose-dependent increases of arterial pressure and heart rate that were not seen with injections adjacent to the dorsal motor nucleus of the vagus. The responses to glycine were neurally mediated and could be blocked either by local pretreatment with strychnine or by combined vagotomy and ganglionic blockade. The data suggest that glycine receptors on, or in the region of, neurons of the dorsal motor nucleus of the vagus may have a role in the regulation of arterial pressure and heart rate.

Nucleus prepositus hypoglossi. A medullary pressor region.

Electrical stimulation of fibers of passage through the fastigial nucleus increases arterial pressure. To identify nuclei that may project through the pressor region of the fastigial nucleus, we injected the retrograde tracer fast blue unilaterally at confirmed pressor sites in the nucleus. In seven rats, we found dense fluorescent labeling bilaterally in the external cuneate, lateral reticular, medial vestibular, and caudal prepositus hypoglossi nuclei, and contralaterally in the inferior olivary nucleus. There had been no reports of a cardiovascular role for the nucleus prepositus hypoglossi; thus, we sought to determine if electrical or chemical stimulation of that nucleus or the adjacent medial vestibular nucleus altered arterial pressure or heart rate in 24 anesthetized rats. Both types of stimuli to the caudal, but not the rostral, pole of the nucleus prepositus hypoglossi or to the medial vestibular nucleus elicited an increase in arterial pressure; bradycardia accompanied the former and tachycardia the latter. Both the nucleus prepositus hypoglossi and medial vestibular nucleus may participate in central cardiovascular regulation.

Vasopressin contributes to hypertension caused by nucleus tractus solitarius lesions.

Lesions of the nucleus tractus solitarius (NTS) were studied to determine whether they elevate plasma vasopressin levels and, if so, whether these elevated levels of vasopressin contribute to the hypertension caused by NTS lesions. Bilateral electrolytic lesions of the NTS caused acute, severe hypertension in rats anesthetized with chloralose and in conscious, freely moving rats. After placement of the NTS lesions there was a greater than tenfold elevation in plasma vasopressin levels. Administration of an antagonist of the vasoconstrictor action of vasopressin markedly diminished the hypertension in both conscious and anesthetized rats. Following ganglionic blockade with chlorisondamine, NTS lesions still elicited hypertension, and the magnitude of the hypertension was not different from that observed in rats not treated with chlorisondamine. The hypertension produced by lesions of the NTS in ganglionic-blocked rats was completely abolished by administration of a vasopressin antagonist. These results indicate that (1) NTS lesions elevate plasma vasopressin levels and (2) elevated plasma vasopressin contributes to the hypertension produced by such lesions.

Mechanisms of cardiovascular responses to glycine injected into the dorsal vagal motor nucleus in rat.

Microinjection of glycine into the dorsal vagal motor nucleus of anesthetized rats elicits increases in arterial pressure and heart rate. In the nucleus tractus solitarii, where cardiovascular responses to injection of glycine may be mediated through release of acetylcholine, there is a dense concentration of glycinergic nerve terminals and glycine receptors. In this study, using immunohistochemical methods, we show that glycine terminals and receptors are present in caudal dorsal vagal motor nucleus, although the concentration of both terminal elements is less than in adjacent nucleus tractus solitarii. Responses to glycine microinjected into the dorsal vagal motor nucleus are blocked by the muscarinic antagonist atropine microinjected at the same site; but, unlike responses to glycine in the nucleus tractus solitarii, responses to glycine in the dorsal vagal motor nucleus are not prolonged by physostigmine. These data support the possibility that endogenous glycine may play a role as a transmitter in the dorsal vagal motor nucleus. Responses to glycine may be mediated through actions at muscarinic receptors but not through acetylcholine itself.

Hemodynamic effects elicited by stimulation of the nucleus tractus solitarii.

Microinjection of the excitatory amino acid L-glutamate into the nucleus tractus solitarii (NTS) elicits decreases in arterial pressure and heart rate. In the present study, we sought to determine the regional hemodynamic effects that were correlated with changes in arterial pressure and heart rate produced by stimulation of the NTS. In anesthetized rats, blood flow in the renal (RBF), superior mesenteric (MBF), and hindquarter (HBF) vascular beds was measured by pulsed Doppler flowmeters. Relative vascular resistances (RVR, MVR, and HVR) were calculated by dividing mean arterial pressure (mm Hg) by the Doppler shift (kHz). Microinjection of L-glutamate into the NTS caused rapid, transient, dose-related decreases in mean arterial pressure and heart rate. MVR and RVR were minimally changed immediately after injections, but both demonstrated delayed dilatation. In contrast, HVR fell immediately but demonstrated delayed constriction. Identical changes occurred in intact rats and in those with interruption of the baroreflex by sinoaortic denervation. Ganglionic blockade with hexamethonium abolished virtually all L-glutamate-induced responses. This study suggests that NTS neurons exert differential effects on renal, mesenteric, and hindquarter vascular beds and that glutamate-induced regional hemodynamic changes are mediated predominantly through autonomic pathways.

Role of endogenous carbon monoxide in central regulation of arterial pressure.

We investigated the contribution of neural mechanisms to the arterial pressure increase produced by zinc deuteroporphyrin 2,4-bis glycol (ZnDPBG), an inhibitor of endogenous carbon monoxide synthesis. The arterial baroreceptor reflex control of heart rate was examined in rats with and without ZnDPBG pretreatment (45 micromol/kg IP) by analysis of the arterial pressure-heart rate relationship during infusions of phenylephrine or sodium nitroprusside to vary arterial pressure. ZnDPBG increased arterial pressure from 110 +/- 3 to 126 +/- 2 mm Hg without eliciting bradycardia. The maximum gain of the heart rate response to changes in arterial pressure was attenuated by ZnDPBG treatment (-1.9 +/- 0.3 versus -4.8 +/- 1.0 bpm/mm Hg). The possibility that ZnDPBG elevates arterial pressure by attenuating baroreceptor reflex function was addressed by comparing the pressor response to ZnDPBG (45 micromol/kg IP) in rats with and without sinoaortic denervation. The pressor effect of ZnDPBG was similar in rats with and without arterial baroreceptor deafferentation, implying that the increase in pressure is not simply the consequence of attenuated baroreceptor reflex function per se. The possibility that ZnDPBG increases arterial pressure via an effect on the nucleus tractus solitarii (NTS) also was investigated. ZnDPBG (1 nmol in 100 nL) injected into the NTS of rats increased arterial pressure from 111 +/- 4 to 126 +/- 5 mm Hg, and this effect was reversed by an ipsilateral microinjection of carbon monoxide into the NTS. Accordingly, the pressor effect of ZnDPBG may rely on inhibition of carbon monoxide production in the NTS. This implies that carbon monoxide formed by brain heme oxygenase plays a role in the central regulation of arterial pressure.

A hyperthermic syndrome in two subjects with acute hydrocephalus.

Although intracranial hypertension may cause autonomic disturbances, as well as alterations in the regulation of body temperature, an acute hyperthermic syndrome with autonomic disturbance as a consequence of hydrocephalus has not been described previously. Two subjects presented with such a syndrome, with each of several episodes of acute shunt failure and hydrocephalus. With correction of the hydrocephalus, the autonomic disturbances and fever immediately cleared. Observations from human and experimental studies suggest some potential mechanisms for the development of the syndrome. One of the subjects of this report was being treated with neuroleptics at the time of hospitalization; in him, and potentially in other similar patients, the syndrome could easily be confused with the neuroleptic malignant syndrome. The need for prompt appreciation of the correct diagnosis was emphasized by the rapid clearing of all neurological signs after correction of the shunt malfunction in both of these patients.

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.

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.

Release of glutamate in the nucleus tractus solitarii in response to baroreflex activation in rats.

Release of endogenous aspartate and glutamate from the region of the nucleus tractus solitarii was measured in vitro by perfusion methods and in vivo by microdialysis. Stimulation of the nucleus tractus solitarii with 35 mM potassium in vitro significantly increased extracellular concentrations of aspartate and glutamate. Glutamate and aspartate concentrations also increased with dialysis of 100 mM KCl into the nucleus tractus solitarii in vivo, but only changes in glutamate were significant. Experiments in vivo revealed that activation of the baroreflex by intravenous infusion of phenylephrine significantly increased glutamate in dialysates, while hypoventilation that accompanies baroreceptor activation and may activate chemoreceptors tended to increase aspartate but not glutamate. The demonstration that glutamate, but not aspartate, is released with activation of the baroreflex further supports the hypothesis that glutamate is a neurotransmitter of baroreceptor afferents terminating in the nucleus tractus solitarii.

Glycine-containing terminals in the rat dorsal vagal complex.

The present study examined the distribution of glycine and glycine-receptors in the dorsal vagal complex using pre-embedding immunocytochemistry. Glycine-immunoreactive terminals were present in moderate densities in the medial, intermediate, interstitial, commissural and ventrolateral subnuclei of the nucleus tractus solitarii. The dorsolateral nucleus tractus solitarii and the dorsal vagal motor nucleus contained only very few, scattered glycine-containing terminals. Glycine terminals appeared to be concentrated in regions of the dorsal vagal complex receiving primary vagal afferents, though previous studies have suggested that glycine is not present in these afferents. A conspicuously high concentration of glycine terminals was observed in the medial nucleus tractus solitarii where a population of cholinergic neurons has been identified previously. Ultrastructurally glycine immunoreactivity was principally associated with terminals containing flattened, pleomorphic vesicles and forming symmetrical synaptic contacts, mostly with dendrites. Glycine receptor immunoreactivity was present throughout the dorsal vagal complex with little evidence of subnuclear localization. With electron-microscopic examination, glycine receptor immunoreactivity was associated with dendritic membranes and was associated presynaptically with terminals containing flattened pleomorphic vesicles. Overall, the present data provide evidence consistent with a neurotransmitter role for glycine in the dorsal vagal complex. The presence of glycine in regions of the dorsal vagal complex receiving vagal afferents suggests a prominent role for this neurotransmitter in autonomic regulation.

Glycine receptor (gephyrin) immunoreactivity is present on cholinergic neurons in the dorsal vagal complex.

We previously demonstrated that microinjection of exogenous glycine into the nucleus tractus solitarii of anesthetized rats elicits responses that are qualitatively like those elicited by microinjection of acetylcholine at the same site. The responses to glycine, like those to acetylcholine, are blocked by administration of a muscarinic receptor antagonist and prolonged by administration of an acetylcholinesterase inhibitor. Furthermore, glycine leads to release of acetylcholine from the nucleus tractus solitarii and surrounding dorsal vagal complex. An anatomical framework for interactions between glycinergic and cholinergic neurons was established by studies that identified glycine terminals and receptors in the dorsal vagal complex. The current study investigated the relationship between glycine receptors and neuronal elements that were immunoreactive for choline acetyltransferase in the dorsal vagal complex. Neurons that were immunoreactive for choline acetyltransferase were located in the dorsal motor nucleus of the vagus, hypoglossal nucleus and nucleus ambiguus, and stained cells were also present in medial, intermediate, and ventrolateral subnuclei of the nucleus tractus solitarii. We found that glycine receptors, immunolabeled with an antibody to gephyrin, were present on cholinergic dendrites in the nucleus tractus solitarii. Gephyrin immunoreactivity was also present on dendrites that did not stain for choline acetyltransferase. These data further support the contribution of cholinergic neurons in mediating cardiovascular responses to glycine in the nucleus tractus solitarii.

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.