Adrenergic projection from the caudal part of the nucleus of the tractus solitarius to the parabrachial nucleus in the rat: immunocytochemical study combined with a retrograde tracing method

The presence of an adrenergic projection from the nucleus of the tractus solitarius (NTS) to the parabrachial nucleus (PB) was demonstrated by the immunocytochemistry combined with a retrograde tracing method. Numerous neurons containing both phenylethanolamine N-methyltransferase, a marker for adrenaline, and wheat germ agglutinin-conjugated horseradish peroxidase, a retrograde tracer, were detected in the dorsolateral part of the NTS at the level of the area postrema after injection of the tracer into the dorsal PB.     

Enhancement of respiratory response to carbon dioxide produced by lesioning caudal regions of the nucleus of the tractus solitarius

Bilateral lesions of the caudal region of the nucleus of the tractus solitarius produced a significant enhancement of the ventilatory response to carbon dioxide in awake rats. The result indicates release of an inhibitory influence normally operating at hindbrain level. Respiration in air or oxygen was not affected while hypoxic responses were depressed insignificantly.     

Modulation of brown adipose tissue-mediated thermogenesis by lesions to the nucleus tractus solitarius in the rat

Given that relatively little is known regarding the central control of brown adipose tissue (BAT)-mediated thermogenesis the present study assessed whether the direct pharmacological stimulation of β- or α-adrenergic receptors located on the brown adipocytes would result in a typical thermogenic response following electrolytic lesions to the nucleus tractus solitarius (NTS). Bilateral electrolytic lesions to the NTS in the rat effectively disrupted the baroreceptor reflex arc. It was observed that the metabolic and temperature responses to either norepinephrine (1, 5, or 25 μg/kg/min) or to the β-agonist isoproterenol (0.5 μg/kg/min) were significantly attenuated in the NTS-lesioned rats relative to the control animals with an intact baroreflex. Conversely, the cardiovascular effects of norepinephrine or of the α-agonist phenylephrine (10 μg/kg/min) were enhanced in the NTS-lesioned animals. The results suggest that the functional capacity of the brown adipocytes was reduced following NTS lesions and points to an alteration in the ability of β-receptors to respond to pharmacological stimulation with a typical thermogenic response.     

Rhythmic neuronal activities in the nucleus of the tractus solitarius isolated in vitro

Coronal slices of the rat medulla were used for an electrophysiological study of the nucleus of the tractus solitarius isolated in vitro. Half of the spontaneously active neurons in the ventral NTS exhibited a rhythmic repetitive spike discharge. In contrast, bursting activity was virtually absent in the isolated NTS. Resetting of repetitive discharges could be obtained by electrical stimulation of the glossopharyngeal-vagal afferent terminals in the tractus solitarius. These results are discussed in relation to the proposed role of the NTS in the neurogenesis of the respiratory rhythm.     

Effect of a high-salt diet on gamma-aminobutyric acid-mediated responses in the nucleus tractus solitarius of Sprague-Dawley rats

Previous study using an indirect gamma-aminobutyric acid (GABA) agonist indicated that high salt intake enhances sensitivity of nucleus tractus solitarius (NTS) projecting inhibitory input to rostral ventrolateral medulla sympathoexcitatory neurons. We further investigated the relationship between salt intake and the GABA system in NTS. Sprague-Dawley (S-D) rats consuming high dietary salt (8%) or low dietary salt (0.3%) for 3 weeks were used. Under chloralose-anesthesia, baseline arterial pressure (AP) and heart rate (HR) were similar in both groups. Bilateral injection into NTS of nipecotic acid, GABA(A) receptor agonist (muscimol), or GABA(B) receptor agonist (baclofen) elicited greater pressor responses in high-salt group. GABA(A) receptor antagonist, bicuculline and GABA(B) receptor antagonist, CGP-35348 elicited greater depressor responses. Phenylephrine or nitroprusside (i.v.) elicited similar respective increases or decreases in AP in both groups. Baroreflex sensitivity was similar. Thus, high-salt intake enhances both GABA(A) receptor- and GABA(B) receptor-mediated responses within NTS, thereby inhibiting elevation of AP.     

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.     

Glucose responding neurons in the nucleus tractus solitarius of the rat: In vitro study

Glucose response of 162 neurons in the nucleus tractus solitarius (NTS) was examined in brainstem slices. When glucose was applied by addition to the bath medium or by electrophoresis, approximately 27% of the neurons responded; 20% were glucose-sensitive neurons which decreased in activity and 7% glucoreceptor neurons which increased in activity. Significantly more glucose-sensitive neurons were found in the caudal part than in the rostral part, while non-glucose responding neurons were distributed widely within the NTS. The populations of glucose-sensitive and glucoreceptor neurons were 11% and 5% in the rostral part, and 28% and 8% in the caudal part of the NTS. Intracellular horseradish peroxidase staining of NTS neurons revealed that glucose-sensitive neurons were medium-sized, fusiform or pyramidal cells with many more dendrites than non-responding small oval cells. Specific functional and morphological characteristics of these glucose responding neurons were clarified by this study.     

Specific subnuclei of the nucleus tractus solitarius play a role in determining the duration of inspiration in the rat

Our previous data obtained in the cat suggest that the neurons of the ventrolateral subnucleus of the tractus solitarius (vlNTS) act as an inspiratory off-switch and terminate the inspiratory phase of the respiratory cycle (Berger et al., Eur. J. Pharmacol. 277 (1995) 195–208; Gillis et al., Neurosci. Abstr. 23 (1997) 725). The purpose of the present study was to determine whether inhibition of the region of the vlNTS of the rat using drugs that hyperpolarize, disfacilitate or block both axonal conduction and action potential generation would alter the inspiratory phase of the respiratory cycle. Experiments were conducted in anesthetized, vagotomized and spontaneously breathing rats while monitoring diaphragmatic electromyogram activity. Vagus nerves were sectioned in order to rule out prolongation of inspiration evoked by microinjection of agents into the vlNTS which block excitatory drive from lung afferent inputs. Bilateral microinjection of the inhibitory amino acid γ-aminobutyric acid (GABA) 25 nmol/45 nl produced an immediate prolongation of inspiratory duration (484±18 to 1291±84 ms) and an apneustic pattern of breathing. Other effects observed were a significant shortening of expiratory duration (778±36 to 432±38 ms), rise in blood pressure (83±4 to 108±6 mmHg) and a small but significant increase in heart rate (439±17 to 452±18 beats/min). Bilateral microinjection of the ionotropic glutamate receptor antagonist kynurenic acid (1 nmol) and the Na⁺ channel blocker tetrodotoxin (10 pmol) into the region of the vlNTS consistently produced a similar prolongation of inspiratory duration and an apneustic pattern of breathing. These results support the hypothesis that neurons in the region of the vlNTS promote the transition from inspiration to expiration and function as part of the ‘Inspiratory Off Switch’.     

Collateral input to the paraventricular and supraoptic nuclei in rat. II. Afferents from the ventral lateral medulla and nucleus tractus solitarius

In the rat, medullary afferents to the hypothalamic magnocellular nuclei mediate the baroreceptor reflexes of vasopressinergic neurons and the cholecystokinin- or gastric distention-induced excitation of oxytocinergic neurons. One strategy that reflexes such as these may use to coordinate the activity of magnocellular neuroendocrine neurons is collateral branching of input. Previous work has shown that the distributions of medullary neurons projecting to the paraventricular and the supraoptic nuclei overlap and that their axons branch. Thus, we hypothesized that single neurons in the ventral lateral medulla and/or the nucleus tractus solitarius would project to both the paraventricular and supraoptic nuclei via collateral branches of their axons. Medullary afferent neurons were retrogradely labeled after injection into the paraventricular and the supraoptic nucleus on one side of the brain with two different fluorescent tracers: Fluoro-Gold or rhodamine-labeled latex microspheres. The topographic distribution of labeled cells in the medulla containing either a single fluorescent tracer or both tracers were plotted. Of these labeled neurons, a small percentage (7%) contained both dyes, suggesting that they send collateral branches to both of the magnocellular neuroendocrine nuclei injected. Single labeled cells were both ipsi- and contralateral to the injected side (53% ipsilateral), but most double-labeled cells were ipsilateral (84%). In rats, areas that project to both the paraventricular and the supraoptic nuclei may act upon both nuclei together. Thus, afferent inputs, in conjunction with the known inter- and intracellular changes that take place within the magnocellular nuclei, may be involved with the coordinated responses throughout magnocellular neuroendocrine system during medullary reflexes, i.e., the baroreceptor-mediated reflexes or the gastric distention reflexes.     

A combined immunocytochemical and retrograde tracing study of noradrenergic connections between the caudal medulla and bed nuclei of the stria terminalis

Region-specific noradrenergic inputs to the bed nuclei of the stria terminalis (BST) from the caudal medulla were studied using combined Fast Blue injections and tyrosine hydroxylase immunoreactivity (TH-ir). Injections into the rostral, dorsal, ventral and lateral BST resulted in predominantly ipsilateral retrograde labelling restricted to the mediodorsal and ventrolateral caudal medulla. Mediodorsal projecting neurones comprised the A2 TH-ir and a second non-aminergic group medial to A2. All ventrolateral retrogradely labelled neurones showed TH-ir and corresponded to A1. Injections into the caudal BST did not label the A2 and very few A1 neurones, indicating a paucity of noradrenergic inputs from this area of the medulla.     

Substance P innervation of neurons projecting to the paraventricular hypothalamic nucleus in the rat nucleus tractus solitarius

After injection of WGA-HRP-colloidal gold in the rat paraventricular nucleus (PVN), retrogradely labeled neurons were found mainly in the medial and commissural subnuclei of the nucleus tractus solitarius (NTS) around 0.5 mm caudal to the obex which is closely related to cardiovascular function. Electron microscopic immunohistochemistry in these areas demonstrated synaptic contacts between retrogradely labeled neurons and substance P-immunoreactive terminals. Innervation of NTS-PVN projection systems by substance P is suggested.     

Distribution of neuropeptide immunoreactive nerve terminals within the subnuclei of the nucleus of the tractus solitarius of the rat

The location of substance P, enkephalin and somatostatin (SRIF), and neurophysin II immunoreactive nerve terminals and preterminal processes in the caudal part of the nucleus of the tractus solitarius (nTS) was examined by the indirect immunofluorescence method for immunocytochemistry combined with cytoarchitectural identification of nuclear subgroups in the same tissue. In 22 Sprague-Dawley rats we examined 14-micrometers-thick serial sections of the dorsal medulla at levels from 1 mm caudal to 2 mm rostral to the obex. These sections were incubated with substance P, enkephalin, somatostatin, and neurophysin II antisera. All four peptides were examined in each case and five typical levels (two caudal and three rostral to the obex) were selected for comparison of terminal distribution between peptides. All sections were photographed under the fluorescence microscope and then counter-stained with cresyl violet. This method of analysis revealed distinct patterns of neuropeptide immunoreactivity in the subnuclei of the nTS that varied according to the level of the section. The nTS is responsible for integrating respiratory, cardiovascular (baroreceptor and cardiac), and gastrointestinal functions. The ventrolateral subnucleus (Vl)nTS, ventral subnucleus (v)nTS, interstitial subnucleus (ni)nTS, and intermediate subnucleus (nI)nTS are the major respiratory subnuclei with vlnTS and vnTS prominently associated with pulmonary afferents, ni associated with laryngeal afferents, and nI with tracheal afferents. The vlnTS, vnTS, and ni showed a moderate density of somatostatin-positive nerve terminals, scattered substance P and enkephalin immunoreactivity, and no neurophysin II-positive terminals. The nI showed moderate density of substance P immunoreactive nerve terminals. The subnuclei of the nTS receiving baroreceptor and chemoreceptor afferents--dorsolateral and dorsal (dl and d) subnuclei of nTS--showed scattered substance P immunoreactive nerve terminals. The commissural nucleus of nTS (ncom), which receives most of the cardiac afferents, showed a moderate density of enkephalin-positive immunoreactive nerve terminals. The medial subnucleus (m)nTS at levels rostral to the obex, the primary site for the termination of gastrointestinal afferents, showed substance P immunoreactivity in moderate amounts and weak immunoreactivity for all the other neuropeptides. An important result of these experiments was the observation that regions of the medulla adjacent to the nTS, i.e., the ventral parasolitarius region (vPSR), dorsal (d)PSR, and the periventricular region (PVR) showed the densest amounts of immunoreactive nerve terminals.(ABSTRACT TRUNCATED AT 400 WORDS)     

Arginine-vasopressin in nucleus of the tractus solitarius induces hyperglycemia and brain glucose retention

Hypothalamic arginine-vasopressin (AVP) plays an important role both as a neurotransmitter and hormone in the regulation of blood glucose and feeding behavior. AVP-containing axons from the parvocellular subdivision of paraventricular nucleus of the hypothalamus terminate in the nucleus of the tractus solitarius (NTS), but the function of this projection is not known. Interestingly, the NTS also receives afferent information from the carotid body and other peripheral receptors involved in glucose homeostasis. We have previously reported that stimulation of the carotid body receptors initiates a hyperglycemic reflex and increases brain glucose retention. Here we show that direct administration of micro-doses of AVP into the NTS of anesthetized or awake rats rapidly increased the levels of blood glucose concentration and brain arterio-venous (A-V) glucose difference. This effect was not observed when the same doses of AVP were injected in the brainstem outside NTS. Arginine-vasopressin antagonist microinjections alone produced a small but significant reduction in brain A-V glucose. Pre-administered VP1-receptor antagonist [beta-mercapto-beta,beta-cyclopentamethylene-propionyl(1),O-Me-Tyr(2),Arg(8)]vasopressin blocked the effects of AVP. These results indicate that AVP acting on its receptors locally within the NTS participates in glucose homeostasis, increasing both blood glucose concentration and brain A-V glucose differences. Hypothalamic AVP may facilitate hyperglycemic responses initiated by peripheral signals processed at the level of the NTS.     

Distribution of neurophysin II immunoreactive nerve fibers within the subnuclei of the nucleus of the tractus solitarius of the rat

The location of neurophysin II immunoreactive nerve fibers and preterminal processes has been examined in various functionally distinct subnuclei of the nucleus of the tractus solitarius (nTS) using the indirect immunofluorescence method for immunocytochemistry combined with cytoarchitectonic identification. The nTS is responsible for integrating respiratory and autonomic reflex activity: the vlnTS, vnTS, ni and nI are associated with respiratory activity; the dlnTS and dnTS are important sites for the integration of baroreceptor and chemoreceptor activity; the ncom, dnTS and dlnTS integrate cardiac afferent activity and the mnTS mediates both cardiovascular and gastrointestinal effects. At levels caudal to the obex, the ncom contained the largest number of neurophysin II immunoreactive nerve fibers and the mnTS and dmnX contained moderate neurophysin II immunoreactivity. At levels rostral to the obex the region of the dorsal medulla adjacent to the mnTS and dnTS (PVR and dPSR) showed the densest immunoreactivity and the mnTS, dmnX and vPSR showed moderate immunoreactivity. At the rostral pole of the nTS, neurophysin II immunoreactive nerve terminals were seen in the dendritic regions of cells in dmnX and mnTS. This selective distribution of neurophysin II immunoreactive nerve terminals in the cardiovascular and gastrointestinal subnuclei of the nTS implicates a direct, descending, hypothalamic, oxytocin-neurophysin II containing pathway interacting with these nTS functions. These results confirm the hypothesis (Sawchenko and Swanson) that descending neurophysin II immunoreactive pathways represent an important neuronal system for the hypothalamic regulation of cardiovascular (vasomotor) and gastrointestinal nuclei in the brainstem.     

Local axonal arborization patterns of distinct neuronal types in the caudal nucleus of the tractus solitarius

Neurons in the caudal nucleus of the tractus solitarius (cNTS) are quite heterogeneous in cell size (50 to 450 microm(2) in somal area) and other morphologic characteristics. For a more objective classification of cNTS neurons, their morphologic features were analyzed quantitatively based on reconstructed biocytin-filled cells after whole-cell patch-clamp recordings. According to the patterns of axonal branching behaviors, cNTS cells could be classified into two groups: smaller cells (94.1 microm(2) in mean somal area, range 62-120 microm(2), n = 22) and larger cells (245 microm(2) in mean somal area, range 142-411 microm(2), n = 23). Extensive axonal arborization with numerous possible synaptic boutons was specifically associated with smaller neurons, while larger cells possessed no or few axon collaterals, suggesting their distinct roles as local circuit neurons (or interneurons) and projection neurons, respectively. With regard to somatodendritic characteristics, the following correlations with cell size were found: smaller cells had larger form factors than larger cells (P < 0.05). Larger neurons had more extensive dendritic arborization, expressed by total dendritic length (P < 0.01) and number of dendritic branching points (P < 0.01), than smaller cells. It was suggested that small cNTS neurons contribute specifically to an integration of input information generated in the local circuits, while large neurons convey the integrated information to other autonomic brain regions.     

Substance P-like immunoreactive axon varicosities with synaptic junctions in the nucleus tractus solitarius of the rat

Substance P-like immunoreactive axon varicosities form both Gray's type I and type II synaptic junctions to the dendrites and somata in the nucleus of the tractus solitarius of the rat. Peroxidase anti-peroxidase reaction products are observed mainly on the large dense-cored vesicles and in the axoplasm preferentially surrounding small clear vesicles. Labeled axon varicosities are filled with small clear vesicles of 50 nm in diameter.     

Mapping of carotid baroreceptor subtype projections to the nucleus tractus solitarius using c-fos immunohistochemistry

This study has combined physiological pressure stimulation of carotid baroreceptors via a vascularly isolated carotid sinus and anodal block of baroreceptor afferent fibers in the carotid sinus nerve to examine the medullary projections of type I vs. type II (large A- vs. small A- and C-fiber afferent) baroreceptors. The control distribution of cells in the nucleus tractus solitarius expressing c-fos in response to physiological activation of carotid baroreceptors in the isolated sinus was compared to that during anodal block of large A-fibers in the carotid sinus nerve. Carotid baroreceptor stimulation primarily activated neurons in the ipsilateral commissural and medial subnuclei of the caudal nucleus tractus solitarius and the dorsolateral, dorsomedial and medial subnuclei in the intermediate and rostral levels of the nucleus tractus solitarius. Elimination of large A-fiber carotid baroreceptor afferents, during similar carotid baroreceptor stimulation resulted in a decrease in the nmber of cells expressing c-fos in the dorsomedial subnucleus of the rostral nucleus tractus solitarius. These data indicate that projections of larger A-fiber (type I) carotid baroreceptors are localized primarily to the rostral dorsomedial subnucleus, while those of smallr A- and C-fiber baroreceptors are more widely distributed to the commissural, medial and dorsal subnuclei of the nucleus tractus solitarius.     

Substance P and serotonin mutually reverse their excitatory effects in the rat nucleus tractus solitarius

Actions and interactions of serotonin and substance P are described in the nucleus tractus solitarius using coronal brainstem slices and intracellular recordings. Substance P (10-100 nM) and serotonin (10-100 microM) applied alone were excitatory, causing depolarization and increasing the input resistance. Reversing effect was obtained using a protocol of long (greater than 5 min) conditioning applications of substance P and shorter (20-60 s) test applications of serotonin: serotonin, which was excitatory by itself during controls, became inhibitory for the steady action potential discharges induced by conditioning substance P applications. In the reverse situation, inhibition was also obtained using prolonged conditioning exposures to serotonin and test applications of substance P. Prolonged conditioning applications (greater than 5 min) were required in these experiments since addition or potentiation, but not inhibition, was found when combining 20-60 s substance P and serotonin applications. In addition to their excitatory effects, substance P and serotonin, applied alone, had another mechanism of action. They reduced the duration of tetraethylammonium-prolonged action potentials. This mechanism was also reversed using conditioning applications of substance P or serotonin. Thus, reversing effects appeared simultaneously on multiple ionic mechanisms. Furthermore, the reversing effect was unaffected in tetrodotoxin-treated preparations, which indicates a postsynaptic phenomenon. Consequently, the control of two different postsynaptic ionic mechanisms during substance P and serotonin interaction suggests that the underlying mechanisms take place at a common level, possibly in relation to second messenger processes. From a functional point of view, these results support the idea that in the nucleus tractus solitarius the effects of either neurotransmitter, serotonin or substance P, can be completely reversed by a previous release of the other one.     

Synaptic contacts of substance P-immunoreactive axon terminals in the nucleus tractus solitarius onto neurons projecting to the caudal ventrolateral medulla oblongata in the rat

The possibility that substance P (SP)-immunoreactive axon terminals in the nucleus tractus solitarius (NTS) make synaptic contacts onto NTS neurons projecting to the catecholaminergic cell region in the caudal ventrolateral medulla oblongata (CVLM) was examined in the rat using a retrograde tract-tracing method combined with immunohistochemistry. After injection of a retrograde tracer, wheat germ agglutinin-conjugated horseradish peroxidase-colloidal gold complex (WGA-HRP-gold), into the CVLM region where tyrosine hydroxylase-immunoreactive neurons were situated, many retrogradely labeled neurons were detected in the dorsal parts of the NTS, especially at levels between 1.0 mm caudal and 0.5 mm rostral to the obex. Immunoelectron microscopy revealed synaptic contacts between SP-immunoreactive axon terminals and WGA-HRP-gold-labeled neurons in the NTS. These findings indicated that SP regulates NTS neurons which project to the catecholaminergic cell region of the CVLM. ©1997 Elsevier Science B.V. All rights reserved.