Projections from the nucleus and tractus solitarius in the cat

After lesions in the nucleus of the tractus solitarius (NTS) in cats and kittens, the termination of degenerating fibers was localized using the Nauta and the Fink-Heimer techniques. The distribution of degenerating fibers was compared with that seen after lesions of the dorsal nucleus of the vagus (DNV) and after section of the ninth and tenth cranial nerves. The projection from the NTS is to the nucleus ambiguus (A), the other divisions of the NTS including the medial NTS and the ventrolateral NTS, the DNV, the medial reticular formation ventral to the NTS (probably the paramedian reticular nucleus), the nucleus intercalatus (INC), and the intermediate nucleus (INT). The probable functional significance of projections from the NTS to these medullary nuclei is discussed in relation to pathways of cardiovascular reflexes. Of particular note is the projection to the INT which may be part of a descending pathway to spinal cardiovascular neurons in the intermediolateral horn.     

Cytochrome oxidase activity in the nucleus of the tractus solitarius of the cat

We studied the cytochrome oxidase (CO) activity in the nucleus of the tractus solitarius (NTS) of normal cats and in animals subjected to unilateral removal of vagal and glossopharyngeal afferents. In normal cats CO activity was higher in the ventrolateral, dorsolateral, interstitial and ventral NTS subnuclei. The dorsal, medial, commissural and gelatinosus subdivisions showed lower levels of CO activity. The peripheral deafferentation up to 47 days did not reduce the CO activity, suggesting an important role for the central inputs in determining the neural activity of the NTS.     

The distribution of glutamate, GABA and aspartate in the nucleus tractus solitarius of the cat

This report describes the distribution of glutamate, GABA and aspartate in the nucleus tractus solitarius (NTS), area postrema, dorsal vagal nucleus and hypoglossal nucleus. Glutamate levels were particularly high in the dorsal, medial and commissural nuclei of the NTS. Section of the IXth and Xth cranial nerves caused a bilateral decrease in glutamate (ipsilateral side = 48%; contralateral side = 36%) and ipsilateral decrease in GABA (27%) in the dorsal and medial NTS. The ipsilateral dorsal vagal nucleus showed a 48% decrease in glutamate and a 30% decrease in GABA after axotomy.     

Catecholamine inputs to the nucleus tractus solitarius

The projections of brainstem catecholaminergic (CA) cell groups to the rat nucleus tractus solitarius (NTS) were examined using 6-hydroxydopamine (6-OHDA) injections and glass microknife cuts. 6-OHDA (4 micrograms) was injected into the intermediate NTS, and this resulted in depletion of CA fluorescent varicosities from the NTS at this rostrocaudal level, except for varicosities along the periventricular edge of the NTS. In addition, a band of swollen fluorescent axons extended between the CA A1 cell group of the ventrolateral medulla and the lateral NTS. Microknife cuts were used to interrupt the projections of the CA A1 and A2 cell groups (located in the caudal NTS) and tissues were examined for changes in CA varicosity density within the intermediate NTS. Following transverse knife cuts of the intermediate NTS, rostral to the A2 cell group, fluorescent varicosities rostral to the cut virtually disappeared, and the fluorescence intensity of the ipsilateral A2 neurons caudal to the cut was increased. These cuts also eliminated the 6-OHDA-resistant varicosities along the periventricular NTS. After microknife cuts lateral to the intermediate NTS, the fluorescent varicosity density in the NTS was unchanged. These results indicate that the major CA projection to the NTS arises from the ipsilateral A2 cell group. The 6-OHDA-resistant varicosities arising from neurons caudal to the knife cut probably arise from the adrenergic C2 cell group.     

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

We describe an intramedullary nitric oxide synthase (NOS) neural pathway that projects from the nucleus tractus solitarius (NTS) to the rostral nucleus ambiguus (NA) in the rabbit. With the use of NADPH diaphorase histochemistry and NOS immunohistochemistry, a compact group of NOS-positive perikarya was identified in the central subnucleus of the NTS dorsomedial to the tractus solitarius and rostral to the obex. A dense network of NOS terminals was seen in the rostral NA. We investigated whether NOS terminals in the NA derive from NOS perikarya in the central NTS and whether the central NOS pathway links esophageal afferents and efferents. In some rabbits, the central NTS was unilaterally lesioned. In others, Phaseolus vulgaris-leucoagglutinin (PHA-L) was injected into the central NTS, or cholera toxin-gold was injected into the NA, or cholera toxin-horseradish peroxidase (HRP) was injected into the wall of the esophagus. The medulla was subsequently processed to demonstrate PHA-L, cholera toxin-gold, HRP, and NOS reactivity. Seven days after the NTS lesion, we observed a marked decrease in the density of NOS terminals in the ipsilateral NA. After injection of PHA-L into the central NTS, a dense group of PHA-L fibres was seen in the rostral NA, principally ipsilaterally. Afferent fibres from the esophagus were found around the NOS cell bodies in the central NTS, and many of these NOS neurons were double labeled with cholera toxin-gold after injection of this tracer into the NA. NOS terminals were found around NA neurons that were retrogradely labelled from the esophagus. We conclude that the NOS neurons in the central NTS act as interneurons in a central pathway connecting esophageal afferents and efferents. © 1995 Wiley-Liss, Inc.     

Behavioural responses to electrical stimulation of the nucleus of the tractus solitarius of the cat

Electrical stimulation of the region of the nucleus tractus solitarius (NTS) significantly reduced both the lever-pressing rate for substantia nigra self-stimulation and the food-consumption in food-deprived cats. In a shuttle box the cats showed no tendency toward shuttling in response to NTS stimulations with randomly varied stimulus intensities. In contrast with these effects, stimulation to nucleus reticularis paramedianus yielded an aversive effect.     

Immunohistochemistry of γ-aminobutyric acid in the cat nucleus tractus solitarius

Using a new antibody directed solely against the γ-aminobutyric acid (GABA) molecule, distribution of GABA was studied in the nucleus tractus solitarii of the cat. Both immunoreactive puncta and cell bodies had a homogenous distribution within the nucleus. The one exception was in the parvocellular subdivision where very little immunoreactive puncta, but numerous immunoreactive cell bodies, were found. Results of this investigation provide immunohistochemical evidence of GABA's localization in an autonomic nucleus involved in cardiovascular regulation.     

Morphology of inspiratory neurons located in the ventrolateral nucleus of the tractus solitarius of the cat

The morphology of 11 dorsal respiratory group (DRG) inspiratory neurons located in the ventrolateral nucleus of the solitary tract (vl-NTS) was studied using the technique of intracellular labeling with the enzyme horseradish peroxidase (HRP). Six of these cells were cut in the transverse plane and had a mean somal diameter of 30.4 m?m, while five others sectioned in the horizontal plane had a mean of 38.2 m?m. These neurons produced an average of 6.2 primary dendrites (range: 4–10), many of which projected rostrally or caudally up to 1.0 mm from the cell bodies. These dendrites were oriented along the longitudinal axis; they ran parallel and ventral to the tractus solitarius. In general, all dendrites possessed numerous spines and appendages. Many axons could be traced for considerable distances within the medulla (in one instance, up to 8 mm). These axons were last discerned in the contralateral ventral medulla rostral to the level of their cell bodies. The axons of three neurons bifurcated in the ipsilateral medulla; one branch remained ipsilateral and projected caudally, while the other crossed the midline. A small number of counterstained cells of size similar to or larger than the HRP-stained neurons formed a column that constituted the vl-NTS. Based upon our observations of stained and counterstained cells, we conclude that the inspiratory neurons of the vl-NTS are few in number and represent a morphologically homogeneous population. The primary orientation of the dendritic arbors of vl-NTS inspiratory neurons appears to optimize the surface area available to receive synaptic contacts from sensory afferents emerging from the tractus solitarius.     

Naturalistic activation of barosensitive afferents release substance P in the nucleus tractus solitarius of the cat

The role for substance P (SP) in baroreceptor transmission in the nucleus tractus solitarius (NTS) remains an area of active research. The purpose of the present study was to determine whether naturalistic activation of barosensitive afferent fibers in the glossopharygneal and vagus nerves release SP in the caudal NTS. Experiments were performed on chloralose anesthetized, artificially ventilated and paralyzed cats. A microdialysis probe was stereotaxically positioned unilaterally in the NTS. Dialysate samples were collected and SP-like immunoreactivity (SP-LI) was measured by radioimmunoassay. Barosensitive afferents were mechanically activated by inflation of a balloon catheter positioned in the thoracic aorta at heart level. Graded balloon inflation produced increases in mean arterial pressure (MAP) of 33±5 mmHg and 60±3 mmHg (P<0.05) and evoked proportional baroreflex decreases in heart rate of 8±3 b.p.m. and 19±3 b.p.m. (P<0.05). This was accompanied by increases in SP-LI of 16±3% and 39±8%, respectively (P<0.05). A positive linear relationship was found between changes in MAP and SP-LI (slope=1.73 fmol/μl/mmHg, r²=0.62) that was completely abolished following barodenervation. These findings provide evidence that naturalistic activation of pressure-sensitive afferents in the glossopharygneal and vagus nerves release SP in a region of the NTS that receives primary afferent projections from aortic, carotid sinus and cardiac receptors in the cat.     

Cortical projections to the nucleus of the tractus solitarius: An HRP study in the cat

Recent evidence suggests that autonomic reflexes involving sensations such as olfaction and gustation may be cortically mediated via centripetal pathways to brainstem autonomic centers. A study was therefore undertaken to elucidate one of these pathways in greater detail. Lectin conjugated horseradish peroxidase was injected into the nucleus tractus solitarius. Following standard light microscopic histochemical procedures to reveal horseradish peroxidase activity, the distribution of retrogradely labeled neurons in the cortex was recorded. Retrogradely labeled somata were seen bilaterally in layer five of the orbital gyrus, anterior insular cortex and infralimbic cortex. In other cats, the same tracer was injected into the orbital gyrus or anterior insular cortex. Bilateral anterograde labeling was seen in various subnuclei throughout the rostrocaudal extent of the nucleus tractus solitarius, but was heaviest in rostral regions of the nucleus. Labeling was also seen bilaterally in the spinal trigeminal nucleus. The projection to the nucleus tractus solitarius could allow for cortical modulation of gustatory and visceral information which is conveyed to the brainstem via the facial, glossopharyngeal and vagus nerves.     

Gustatory functions of the nucleus tractus solitarius in the rabbit

Multiunit analysis revealed a rostral region of NTS containing cells responsive to taste stimulation of rostral tongue. Using representative stimuli for the 4 basic types of taste, maximal incidence and magnitude of response was found to NaCl, followed by HCl, sucrose and QHC1. Further analysis of temporal patterns of response to the tastants revealed differences among stimuli in latency and time course. A principal components analysis indicated that time course, apart from magnitude of response, could contribute to neural differentiation of tastants. Information was also derived on neural intensity functions for these basic types of tastants. Additional observations with sodium saccharin revealed minimal neuronal responsivity despite reported evidence of behavioral preference for this tastant by rabbits.     

Excitation of upper cervical inspiratory neurons by inspiratory neurons of the nucleus tractus solitarius in the cat

Experiments were conducted to investigate the connection from inspiratory neurons in the ventrolateral region of the nucleus tractus solitarius to the inspiratory neurons in the contralateral upper cervical (C1-C2) spinal cord. Microstimulation within the region usually produced long-latency (10 to 30 ms) synaptic activation of the upper cervical inspiratory neurons. Antidromic mapping of axons of the ventrolateral nucleus tractus solitarius in C1 demonstrated the existence of collateral arbors in the region of the upper cervical inspiratory neurons (7 of 15). Cross-correlation histograms showed the existence of short-term synchronization of firing for 12 of 74 pairs of ventrolateral nucleus tractus solitarius and upper cervical inspiratory neurons. Cross-correlation combined with antidromic activation showed that the short-term synchronization was due to a direct connection in two cases and a common input in one case. It was concluded that at least some ventrolateral nucleus tractus solitarius inspiratory neurons may directly excite upper cervical inspiratory neurons, probably via monosynaptic connections. Both groups of neurons may also share common inputs.     

Neurochemical transmission of baroreceptor input in the nucleus tractus solitarius

Baroreceptor activation has been found to produce different types of discharge patterns in neurons in the nucleus tractus solitarius (NTS). The contribution of different glutamate receptor subtypes, neuropeptide modulators and input from different baroreceptor subtypes to the generation of firing patterns in NTS barosensitive neurons was examined in a series of studies. Results from these studies indicate that both subtypes of ionotropic glutamate receptors contribute to discharge in barosensitive neurons, and the role of each subtype can vary for different neurons. The neuropeptide neurotensin was found to modulate baroreceptor control of BP and discharge of central barosensitive neurons, both through modulation of baroreceptor afferent input and possibly through release of neurotensin by baroreceptor afferent fibers in the NTS. Finally, selective modulation of input from baroreceptor subtypes indicates that there is some degree of divergent baroreceptor innervation of NTS neurons that could contribute to initiation of their different discharge patterns in response to baroreceptor input.     

Cardiovascular responses to microinjections of nociceptin into a midline area in the commissural subnucleus of the nucleus tractus solitarius of the rat

Immunoreactivity for nociceptin, an endogenous ligand for the ORL1 opioid receptors, has been reported in the nucleus tractus solitarius (nTS). A midline area in the commissural subnucleus (nCom) of nTS is the site of peripheral chemoreceptor projections. This investigation was carried out in urethane-anesthetized, artificially ventilated, adult male Wistar rats, to study the cardiovascular effects of the activation of ORL1 receptors in a midline area of the nCom. Microinjections (30 nl) of nociceptin (0.15-0.62 mM) into the nCom elicited depressor and bradycardic responses. Prior microinjections of [N-Phe(1)]-nociceptin-(1-13)-NH(2) (4.5 mM), a specific antagonist for ORL1 opioid receptors, into the nCom blocked the effects of nociceptin (0.31 mM, the maximally effective concentration), but not endomorphin-2 (0.6 mM; an endogenous ligand for micro -opioid receptors). On of other hand, naloxone (0.125 mM; an antagonist for classical opioid receptors) did not block the effects of nociceptin, while it did block the effects of endomorphin-2. The blockade of nociceptin effects by [N-Phe(1)]-nociceptin-(1-13)-NH(2) and endomorphin-2 by naloxone, was not due to some nonspecific effects because the responses to L-Glu (5 mM) remained unaltered after the microinjection of these antagonists. These results indicate that activation of ORL1 receptors in the nCom may play a role in the regulation of cardiovascular function.     

Strategies for cellular identification in nucleus tractus solitarius slices

The indistinct regional anatomy and intermixing of second order neurons with projection and interneurons make cellular studies more difficult within the nucleus tractus solitarius (NTS). Here, we outline experimental strategies to join in vitro electrophysiological with neuroanatomical protocols to discriminate specific subpopulations of NTS neurons. Horizontally cutting the brain stem produces slices in which electrical activation of the solitary tract (ST) is free of local interneuron contamination. Such ST excitatory synaptic currents (EPSCs) functionally identify second order NTS neurons by their minimal variation of latency (jitter). Sapphire blades, cold cutting temperatures and a mechanically stable microtome were critical to consistently obtain viable slices that were optimized for infrared and fluorescence microscopy. Anterogradely transported carbocyanine dye implanted on the aortic depressor nerve anatomically identified second order NTS neurons and their ST synaptic performance conformed to the minimal jitter signature of second order neurons. Retrograde tracers and green fluorescent protein labeled neurons afford two additional promising approaches for discriminating NTS neuron phenotypes in broader system contexts. Detailed methods and troubleshooting are described. Coupling tracing techniques with electrophysiology adds important new dimensions to NTS studies and such strategies provide bridging information between cellular mechanisms, neuroanatomy and systems integration.     

Segregation of coarse and fine glossopharyngeal axons in the visceral nucleus of the tractus solitarius of the cat

The projections of coarse and fine axons of the glossopharyngeal (IX) nerve upon the caudal two thirds of the nucleus of the tractus solitarius (NTS) were studied in the cat. These afferents convey the chemo- and baroreceptor activities from the carotid receptors. We applied the Fink-Heimer method on brainstem sections, at different survival times, after a petrosal ganglionectomy. A segregation of fine and coarse fibered components was observed. Degeneration of coarse axons was mostly found in the lateral NTS, while fine fiber degeneration was predominant in regions of the medial and commissural NTS. The injection of WGA-HRP in the different NTS divisions demonstrated that the lateral NTS was mainly innervated by the set of largest neurons of the petrosal ganglion and that the medial and the commissural NTS were innervated by the set of smaller neurons of the ganglia. These results were discussed in relation to cytoarchitecture, myeloarchitecture, distribution of normal axons, and known central connectivity of the different NTS divisions. We concluded that coarse and fine visceral afferents of the IX nerve, which includes the afferents of the carotid body and the carotid sinus, represent different afferent populations that project to particular divisions of the NTS and connect to different central pathways.     

Experimental study of the projections of the nucleus of the tractus solitarius and the area postrema in the cat

Projections from the area postrema and adjacent parts of the medial solitary nucleus are demonstrated with the Nauta method following lesions limited exclusively to these structures. Experiments are controlled with lesions involving adjacent bulbar regions, cerebellum, and spinal cord. Ascending pathways in the dorsal and lateral columns of the spinal cord project ipsilaterally to the area postrema and bilaterally to a para-alar nucleus in the ventral periphery of the nucleus gracilis. Neurons in the area postrema project mainly inspilaterally to the dorsal and medial regions of the medial solitary nucleus. Neurons in the posterior half of the medical solitary nucleus project ipsilaterally to the lateral solitary nucleus, dorsal vagal nucleus, ambigus, retrofacial nucleus, and dorsal and lateral bulbar reticular formation. Projections to nuclei intercalatus and prepositus hypoglossi, bilaterally, and to the ipsilateral dorsal tegmental nucleus by way of the dorsal longitudinal fasciculus are also shown. No direct projections to the diencephalon are demonstrated. Control lesions in the dorsal column nuclei reveal projections to the contralateral inferior olive and thalamic reticular and ventrobasal nuclei, but not to the projection sites of the solitary nucleus. Evidence is given to support the hypothesis that ascening visceral pathways are interruped in the bulbar reticular formation and dorsal tegmental nucleus before reaching the diencephalon. Correlations are suggested with functional aspects of the central autonomic and reticular activating systems.     

Characterization of neurons of the nucleus tractus solitarius pars centralis

Esophageal sensory afferent inputs terminate principally in the central subnucleus of the tractus solitarius (cNTS). Neurons of the cNTS comprise two major neurochemical subpopulations. One contains neurons that are nitric oxide synthase (NOS) immunoreactive (-IR) while the other comprises neurons that are tyrosine hydroxylase (TH)-IR. We have shown recently that TH-IR neurons are involved in esophageal-distention induced gastric relaxation. We used whole cell patch clamp techniques in rat brainstem slices combined with immunohistochemical and morphological reconstructions to characterize cNTS neurons. Postrecording reconstruction of cNTS neurons revealed two morphological neuronal subtypes; one group of cells (41 out of 131 neurons, i.e., 31%) had a multipolar soma, while the other group (87 out of 131 neurons, i.e., 66%) had a bipolar soma. Of the 43 cells in which we conducted a neurochemical examination, 15 displayed TH-IR (9 with bipolar morphology, 6 with multipolar morphology) while the remaining 28 neurons did not display TH-IR (18 with bipolar morphology, 10 with multipolar morphology). Even though the range of electrophysiological properties varied significantly, morphological or neurochemical distinctions did not reveal characteristics peculiar to the subgroups. Spontaneous excitatory postsynaptic currents (sEPSC) recorded in cNTS neurons had a frequency of 1.5 +/- 0.15 events s(-1) and an amplitude of 27 +/- 1.2 pA (Vh = -50 mV) and were abolished by pretreatment with 30 muM AP-5 and 10 muM CNQX, indicating the involvement of both NMDA and non-NMDA receptors. Some cNTS neurons also received a GABAergic input that was abolished by perfusion with 30-50 muM bicuculline. In conclusion, our data show that despite the heterogeneity of morphological and neurochemical membrane properties, the electrophysiological characteristics of cNTS neurons are not a distinguishing feature.