Influence of nucleus tractus solitarius stimulation and baroreceptor activation on rat parabrachial neurons.

The parabrachial nucleus (PBN) within the dorsolateral pons is a major recipient of autonomic-related inputs from more caudal levels of the brainstem and, in particular, the nucleus of the solitary tract (NTS). Although the anatomical projections from the NTS to the PBN are well characterized, less is known concerning the influence of activating NTS efferents on PBN neurons and the response of the latter to cardiovascular-related inputs. The present study examined the response of PBN neurons to electrical stimulation of the depressor area within the NTS in urethane anesthetized rats, and subsequently, the influence of arterial baroreceptor activation and systemic angiotensin II (ANG II) on these cells. Extracellular single-unit PBN recordings indicated that 92 of 227 (40.5%) cells were orthodromically excited and 35 of 227 (15.4%) inhibited consequent to NTS stimulation. Ten (4.5%) PBN cells displayed antidromic activation from the NTS. Of 41 of 119 neurons responding to both NTS stimulation and baroreceptor activation, 29.3% revealed a excitatory and 31.7% an inhibitory response to the two stimuli. Fifteen PBN cells responded to NTS stimulation, baroreceptor activation, and the administration of systemic ANG II, with six cells displaying either an excitatory or inhibitory response to all three stimuli. These observations provide electrophysiological support for reciprocal connections between the NTS and PBN and indicate the presence of both excitatory and inhibitory projections to the pontine nucleus. A population of neurons influenced by activation of NTS efferents also reveal a similarity of responses to inputs originating from peripheral arterial baroreceptors and systemic ANG II.     

Synaptic and morphologic properties in vitro of premotor rat nucleus tractus solitarius neurons labeled transneuronally from the stomach

Neurons in the rat nucleus tractus solitarius (NTS) possess morphologic characteristics that have been correlated with the type of synaptic information they receive. These features have been described for viscerosensory neurons but not for premotor NTS neurons. The morphologic and synaptic features of neurons in the rat caudal NTS were assessed using whole-cell patch-clamp recordings and biocytin labeling in brainstem slices. Gastric-related premotor NTS neurons were identified for recording after inoculation of the stomach wall with a transneuronal retrograde viral label that reports enhanced green fluorescent protein. Three morphologic groups of NTS neurons were identified based on quantitative aspects of soma area and proximal dendritic arborization, measures that were consistent across slice recordings. The most common type of cell (group I) had relatively small somata and one to three sparsely branching dendrites, whereas the other groups had larger somata and more than three dendrites, which branched predominantly close to (group II) or distant from (group III) the soma. Voltage-clamp recordings revealed spontaneous excitatory and inhibitory postsynaptic currents in all neurons, regardless of morphology. Gastric-related premotor NTS neurons composed two of the three morphologic types (i.e., groups I and II). Compared with unlabeled neurons, these cells were less likely to receive constant-latency synaptic input from the tractus solitarius. These results refute the hypothesis that general patterns of synaptic input to NTS neurons depend on morphology. Gastric premotor neurons comprise a subset of NTS morphologic types, the organization of the viscerosensory input to which has yet to be defined.     

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.     

Synaptic responses of neurons of the nucleus tractus solitarius in vitro

Postsynaptic responses of neurons in the nucleus tractus solitarius (NTS) have been studied in an in vitro slice preparation using extra- and intracellular recording. Single or paired pulse stimulations were delivered to afferent fibers within the tractus solitarius (TS) to activate orthodromic responses in these neurons. Most NTS neurons displayed an initial synaptic excitation followed by inhibition of spontaneous or evoked firing lasting up to 150-200 ms after stimulation. Excitatory postsynaptic potentials (EPSPs), recorded intracellularly, were increased in amplitude by membrane hyperpolarization. Large afterhyperpolarizations followed action potentials triggered by the EPSPs or evoked by intracellular current injections. Intracellular evidence for synaptic inhibition within the NTS included: (1) the presence, after Cl-injection, of flurries of spontaneous PSPs likely to be inverted inhibitory postsynaptic potentials; (2) reduction of the size of a test EPSP by a previous subthreshold TS conditioning volley; and (3) hyperpolarizing PSPs recorded in some neurons. Other NTS neurons exhibited prolonged excitatory responses to TS stimulation and could be local inhibitory interneurons. These results may help specify synaptic mechanisms in the NTS that could play an integrative role in the relay of visceral sensory inputs to higher order effectors.     

Substance P immunoreactive nerve terminals in the dorsolateral nucleus of the tractus solitarius: roles in the baroreceptor reflex

Physiological and light microscopic evidence suggest that substance P (SP) may be a neurotransmitter contained in first-order sensory baroreceptor afferents; however, ultrastructural support for this hypothesis is lacking. We have traced the central projections of the carotid sinus nerve (CSN) in the cat by utilizing the transganglionic transport of horseradish peroxidase (HRP). The dorsolateral subnucleus of the nucleus tractus solitarius (dlNTS) was processed for the histochemical visualization of transganglionically labeled CSN afferents and for the immunocytochemical visualization of SP by dual labeling light and electron microscopic methods. Either HRP or SP was readily identified in single-labeled unmyelinated axons, myelinated axons, and nerve terminals in the dlNTS. SP immunoreactivity was also identified in unmyelinated axons, myelinated axons, and nerve terminals in the dlNTS, which were simultaneously identified as CSN primary afferents. However, only 15% of CSN terminals in the dlNTS were immunoreactive for SP. Therefore, while the ultrastructural data support the hypothesis that SP immunoreactive first-order neurons are involved in the origination of the baroreceptor reflex, they suggest that only a modest part of the total sensory input conveyed from the carotid sinus baroreceptors to the dlNTS is mediated by SP immunoreactive CSN terminals. Five types of axo–axonic synapses were observed in the dlNTS. SP immunoreactive CSN afferents were very rarely involved in these synapses. Furthermore, SP terminals were never observed to form the presynaptic element in an axo–axonic synapse with a CSN afferent. Therefore, SP does not appear to be involved in the modulation of the baroreceptor reflex in the dlNTS.     

Non-NMDA receptors in the nucleus of the tractus solitarius play the predominant role in mediating aortic baroreceptor reflexes.

The purpose of these studies was to determine the relative role of N-methyl-D-aspartic acid (NMDA) receptors and non-NMDA receptors in the nucleus of the tractus solitarius (NTS) in mediating arterial baroreceptor reflexes evoked by electrical stimulation of the aortic nerve. Selective blockade of NMDA receptors in the NTS had little effect on aortic baroreflexes except at high frequencies of aortic nerve stimulation. In contrast, blockade of non-NMDA receptors in the NTS abolished aortic baroreceptor reflexes. These results suggest that although NMDA receptors may modulate baroreflex responses, synaptic activation of non-NMDA receptors in the NTS plays the predominant role in mediating aortic baroreceptor reflexes.     

Ultrastructural studies on catecholaminergic terminals and GABAergic neurons in nucleus tractus solitarius of the rat medulla oblongata

Synaptogenesis of catecholamine (CA) boutons in the nucleus tractus solitarius (NTS) was compared between spontaneously hypertensive (SHR) rats and Wistar-Kyoto (WKY) rats at different ages. On the average, there were about 32 CA varicosities per 2200 μm² area of the NTS in both SHR and WKY rats as revealed by glyoxylic acid fluorescence microscopic (FM) morphometric study. The FM analysis indicated that there were no significant changes in the CA varicosity density between SHR and WKY rats.The CA boutons were labeled with 5-hydroxydopamine and appeared to contain small granular vesicles at the electron microscopic (EM) level. A total of 1402 CA boutons were studied in a 540, 000 μm² area of the NTS. The number of CA boutons involved in synaptic contacts vs the number of total CA boutons was used to obtain synaptic frequency which was taken as an index for synaptogenesis. A reduction of approximately 18% and 14% of synaptogenesis of CA boutons was observed in the NTS of SHR rats at 4 weeks (prehypertensive stage) and 12 weeks (early hypertensive stage) of age respectively, as compared to age-matched WKY rats. No significant difference of synaptogenesis of CA neurons was found between SHR and WKY rats at 16 weeks of age, a stage in which hypertension is well established and maintained in SHR rats. These results suggest that CA neurons with fewer synaptic contacts in the NTS may play a more important role in the initiation than in the maintenance of hypertension in the SHR rats.In addition to CA terminals, there were numerous GABAergic cell bodies in the NTS which were identified by immunocytochemistry using antibodies to the GABA synthesizing enzyme,l-glutamate decar☐ylase (GAD). GABAergic dendrites with GAD-positive reaction were often seen to receive several GAD-negative synapses at EM random profiles. In the text, a viewpoint is thus discussed that emphasizes that a synaptic abnormality of CA terminals with fewer synaptic contacts affecting GABAergic neurons may participate in the pathogenesis of hypertension. However, it remains to be determined as to whether or not there is a direct contact between CA boutons and GABAergic dendrites.     

Tonic stimulation of GABAB receptors in the nucleus tractus solitarius modulates the baroreceptor reflex.

Previous studies have indicated that tonic stimulation of GABAB receptors in the nucleus tractus solitarius (NTS) contributes to the regulation of arterial blood pressure (AP). The present studies examined the hypotheses that (1) tonic stimulation of GABAB receptors in the NTS provides a tonic attenuation of the baroreceptor reflex and (2) enhanced stimulation of these GABAB receptors markedly attenuates the baroreceptor reflex resulting in an increase in AP. In chloralose-anesthetized rats electrical stimulation of the aortic depressor nerve elicited frequency-dependent decreases in AP and heart rate (HR). These responses were markedly attenuated, but not eliminated, by injection of the GABAB receptor agonist baclofen into the ipsilateral NTS. In contrast, the GABAA receptor agonist muscimol completely inhibited aortic depressor nerve-evoked responses. Blockade of GABAB receptors in the NTS by local injection of CGP-35348 elicited a dose-dependent decrease in AP, and a dose-dependent blockade of the pressor response elicited by injection of baclofen into the NTS. These results support the hypothesis that GABA acts tonically on GABAB receptors in the NTS to attenuate the baroreceptor reflex, thereby contributing to the regulation of AP.     

Modulation of the carotid baroreceptor reflex by substance P in the nucleus tractus solitarius

Previous studies have shown that administration of substance P (SP) into the nucleus tractus solitarius (NTS) can evoke a depressor response similar to that produced by activation of the arterial baroreceptors. In addition, some studies have suggested that SP increases the reflex responses to activation of baroreceptor input. The present study was performed to determine the effects of SP on the carotid sinus baroreceptor reflex at the level of the NTS by examining the effects of both exogenous SP microinjected into different rostrocaudal locations in the NTS and blockade of the effects of endogenous SP, through the microinjection of a substance P antagonist (SPa; [D-Pro, D-Trp]-substance P). Changes in pressure in an isolated carotid sinus in anesthetized dogs were used to evoke baroreflex changes in arterial blood pressure (BP) before and after microinjection of SP (0.5 microM) or SPa (10 microM) into barosensitive regions of the NTS. Microinjection of SP or its antagonist did not alter baseline, resting BP but did produce significant changes in baroreflex sensitivity. Microinjection of SP into different rostrocaudal regions of the NTS produced different responses, with rostral and caudal NTS microinjections producing significant increases in sensitivity. No effects on baroreflex sensitivity were obtained in response to SP microinjections into the intermediate NTS. Unlike SP, microinjection of the SPa significantly decreased baroreflex sensitivity at all rostrocaudal levels of the NTS. These data demonstrated that SP has the capability to modulate the carotid baroreflex at the level of the NTS and support a physiological role for endogenously released SP.     

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.     

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.     

Inhibitory effect of cholecystokinin octapeptide on neurons in the nucleus tractus solitarius

We investigated the effect of the cholecystokinin octapeptide (CCK8) applied locally to neurons of the nucleus tractus solitarius (NTS). Results demonstrate an inhibitory effect of CCK8 on spike discharges including those related to respiration. It is suggested that CCK8 acts at this level through specific receptor mechanisms since CCK8-induced inhibitions were not reproduced by application of related peptides and were resistant to antagonists of different inhibitory transmitters.     

Stimulation of the ventrolateral medulla inhibits the baroreceptor input to the nucleus tractus solitarius

Extracellular recording experiments were done in urethane- and chloralose-anaesthetized, paralyzed and artificially ventilated rabbits. Thirty-one baroreceptor-sensitive neurones were identified in the region of the nucleus tractus solitarius (NTS) by their excitatory responses to stimulation of the ipsilateral aortic nerve. A conditioning stimulus delivered to the ipsilateral or contralateral rostral ventrolateral medulla (rVLM) inhibited the excitatory responses of 80.0% (19 out of 24) neurones to an aortic test stimulus as early as 3 ms and extending as long as 400 ms after conditioning. The same inhibitory effect was also observed by application of excitant amino acid DL-homocysteic acid (0.2 M, 100 nl) into the ipsi- or contralateral rVLM area in 6 units. In 5 units inhibited by rVLM stimulation, evoked discharges were inhibited by prolonged electrical stimulation of the superficial peroneal nerve (SP) with low intensity and low frequency (0.1-0.3 mA and 5-10 Hz). These results provide the electrophysiological evidence for the suppressing effect of the rVLM on the excitatory responses of NTS neurones to baroreceptor afferent stimulation.     

Multimodal responses of taste neurons in the frog nucleus tractus solitarius

The responses of 216 neurons in the nucleus tractus solitarius (NTS) of the American bullfrog were recorded following taste, temperature, and tactile stimulation. Cells were classified on the basis of their responses to 5 taste stimuli: 0.5 M NaCl, 0.0005 M quinine-HCl (QHCl), 0.01 M acetic acid, 0.5 M sucrose, and deionized water (water). Neurons showing excitatory responses to 1, 2, 3, or 4 of the 5 kinds of taste stimuli were named Type I, II, III, or IV, respectively. Cells whose spontaneous rate was inhibited by taste and/or tactile stimulation of the tongue were termed Type V. Type VI neurons were excited by tactile stimulation alone. Of the 216 cells, 115 were excited or inhibited by taste stimuli (Types I-V), with 35 being Type I, 34 Type II, 40 Type III, 2 Type IV and 4 Type V. The remaining 101 cells were responsive only to tactile stimulation (Type VI). Of those 111 cells excited by taste stimulation (Types I-IV), 106 (95%) responded to NaCl, 66 (59%) to acetic acid, 44 (40%) to QHCl, 10 (9%) to water, and 9 (8%) to warming. No cells responded to sucrose. Of the 111 cells of Types I-IV, 76 (68%) were also sensitive to mechanical stimulation of the tongue. There was some differential distribution of these neuron types within the NTS, with more narrowly tuned cells (Type I) being located more dorsally in the nucleus than the more broadly tuned (Type III) neurons. Cells responding exclusively to touch (Type VI) were also more dorsally situated than those responding to two or more taste stimuli (Types II and III).     

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.     

Stimulation of alpha 2-adrenergic receptors in nucleus tractus solitarius is required for the baroreceptor reflex.

Bilateral injection into the nucleus tractus solitarius (NTS) of the alpha 2-adrenergic receptor antagonist yohimbine produced a dose-related (10-500 pmol) increase in arterial pressure, with a maximal response of approximately 60 mm Hg. Idazoxan, also an alpha 2-adrenergic receptor antagonist, produced a similar response although idazoxan was less potent than yohimbine. The pressor response elicited by these drugs was attenuated by stimulation of adrenergic receptors in the NTS by local administration of either clonidine or tyramine. Doses of yohimbine (200 pmol) or idazoxan (5 nmol) that maximally increased arterial pressure also completely inhibited the depressor and bradycardic responses to electrical stimulation of the aortic depressor nerve. These results indicate that tonic stimulation of alpha 2-adrenergic receptors in the NTS is required for baroreceptor reflex function.     

Tonic stimulation of GABAB receptors in the nucleus tractus solitarius modulates the baroreceptor reflex

Previous studies have indicated that tonic stimulation of GABA_B receptors in the nucleus tractus solitarius (NTS) contributes to the regulation of arterial blood pressure (AP). The present studies examined the hypotheses that (1) tonic stimulation of GABA_B receptors in the NTS provides a tonic attenuation of the baroreceptor reflex and (2) enhanced stimulation of these GABA_B receptors markedly attenuates the baroreceptor reflex resulting in an increase in AP. In chloralose-anesthetized rats electrical stimulation of the aortic depressor nerve elicited frequency-dependent decreases in AP and heart rate (HR). These responses were markedly attenuated, but not eliminated, by injection of the GABA_B receptor agonist baclofen into the ipsilateral NTS. In contrast, the GABA_A receptor agonist muscimol completely inhibited aortic depressor nerve-evoked responses. Blockade of GABA_B receptors in the NTS by local injection of CGP-35348 elicited a dose-dependent decrease in AP, and a dose-dependent blockade of the pressor response elicited by injection of baclofen into the NTS. These results support the hypothesis that GABA acts tonically on GABA_B receptors in the NTS to attenuate the baroreceptor reflex, thereby contributing to the regulation of AP.     

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.     

Sonic hedgehog signalling in neurons of adult ventrolateral nucleus tractus solitarius

The transmembrane receptor Patched (Ptc) mediates the action of the diffusing factor Sonic hedgehog (Shh), which is implicated in establishing morphogenetic gradients during embryonic development. Whereas alteration of Ptc function is associated with developmental abnormalities and brain tumors, its functional activity and roles in the adult brain have yet to be elucidated. Here we describe the complementary pattern of Shh and Ptc expression in the rat dorsal vagal motor nucleus and the ventrolateral nucleus tractus solitarius (vNTS), respectively. Those two interconnected structures regulate the cardiorespiratory function during hypoxia. Bath application of a subnanomolar concentration of aminoterminal Shh protein (ShhN) to a slice preparation of the vNTS induces a rapid decrease in neuronal firing followed by a bursting activity that propagates in the neuronal network. Intracellular current injections show that bursts result from an action on the neuronal membrane electro-responsiveness. Both inhibiting and bursting effects are blocked by the monoclonal Shh antibody 5E1 and may require the Ptc binding site of ShhN. Thus, ShhN acting on specific neuronal sites controls electrophysiological properties of differentiated neurons of the vNTS. We speculate on a retrocontrol of cardiorespiratory signals in the vNTS, by Shh generated in dorsal vagal motoneurons.