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Previously we have shown that stimulation of inhibitory A1 adenosine receptors located in the nucleus tractus solitarii (NTS) attenuates cardiopulmonary chemoreflex (CCR) evoked inhibition of renal, adrenal and lumbar sympathetic nerve activity and reflex decreases in arterial pressure and heart rate. Activation of facilitatory A2a adenosine receptors, which dominate over A1 receptors in the NTS, contrastingly alters baseline activity of regional sympathetic outputs: it decreases renal, increases adrenal and does not change lumbar nerve activity. Considering that NTS A2a receptors may facilitate release of inhibitory transmitters we hypothesized that A2a receptors will act in concert with A1 receptors differentially inhibiting regional sympathetic CCR responses (adrenal>lumbar>renal). In urethane/chloralose anesthetized rats (n=38) we compared regional sympathetic responses evoked by stimulation of the CCR with right atrial injections of serotonin 5HT3 receptor agonist, phenylbiguanide, (1-8μg/kg) before and after selective stimulation, blockade or combined blockade and stimulation of NTS A2a adenosine receptors (microinjections into the NTS of CGS-21680 0.2-20pmol/50nl, ZM-241385 40pmol/100nl or ZM-241385+CGS-21680, respectively). We found that stimulation of A2a adenosine receptors uniformly inhibited the regional sympathetic and hemodynamic reflex responses and this effect was abolished by the selective blockade of NTS A2a receptors. This indicates that A2a receptor triggered inhibition of CCR responses and the contrasting shifts in baseline sympathetic activity are mediated via different mechanisms. These data implicate that stimulation of NTS A2a receptors triggers unknown inhibitory mechanism(s) which in turn inhibit transmission in the CCR pathway when adenosine is released into the NTS during severe hypotension., (© 2013.)

Second order neurons in the nucleus tractus solitarius (NTS) process and integrate the afferent information from arterial baroreceptors with high fidelity and precise timing synaptic transmission. Since 2nd-order NTS neurons receiving baroreceptors inputs are relatively well characterized, their electrophysiological profile has been accepted as a general characteristic for all 2nd-order NTS neurons involved with the processing of different sensorial inputs. On the other hand, the synaptic properties of other afferent systems in NTS, such as the peripheral chemoreceptors, are not yet well understood. In this context, in previous studies we demonstrated that in response to repetitive afferents stimulation, the chemoreceptors 2nd-order NTS neurons also presented high fidelity of synaptic transmission, but with a large variability in the latency of evoked responses. This finding is different in relation to the precise timing transmission for baroreceptor 2nd-order NTS neurons, which was accepted as a general characteristic profile for all 2nd order neurons in the NTS. In this brief review we discuss this new concept as an index of complexity of the sensorial inputs to NTS with focus on the synaptic processing of baro- and chemoreceptor afferents., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Inhibitory neurotransmission has an important role in the processing of sensory afferent signals in the nucleus of the solitary tract (NTS), particularly in spontaneously hypertensive rats (SHR). In the present study, we tested the hypothesis that γ-aminobutyric acid (GABA) mediated neurotransmission within the NTS produces an inhibition of the baroreflex response of splanchnic sympathetic nerve discharge (sSND). In urethane-anesthetized, artificially ventilated and vagotomized male SHR and Wistar Kyoto (WKY) rats we compared baroreflex-response curves evoked after bilateral injections into the NTS of the GABA-A antagonist bicuculline (25pmol/50nl) or the GABA-B antagonist CGP 35348 (5nmol/50nl). Baseline MAP in SHR was higher than the WKY rats (SHR: 153±5, vs. WKY: 112±6mm Hg, p<0.05). Bilateral injection of bicuculline or CGP 35348 into the NTS induced a transient (5min) reduction in MAP (∆=-26±4 and -41±6mm Hg, respectively vs. saline ∆=+4±3mmHg, p<0.05) and sSND (∆=-21±13 and -78±7%, respectively vs. saline: ∆=+6±4% p<0.05). Analysis of the baroreceptor curve revealed a decrease in the lower plateau (43±11 and 15±5%, respectively vs. saline: 78±6%, p<0.05) and an increase in the sympathetic gain of baroreflex (6.3±0.3, 7.2±0.8% respectively vs. saline: 4.2±0.4%, p<0.05). Bicuculline or CGP35348 into the NTS in WKY rats did not change MAP, sSND and sympathetic baroreflex gain. These data indicate that GABAergic mechanisms within the NTS act tonically reducing sympathetic baroreflex gain in SHR., (Crown Copyright © 2010. Published by Elsevier B.V. All rights reserved.)

In prior studies, we found that activation of cannabinoid-1 receptors in the nucleus tractus solitarii (NTS) prolonged baroreflex-induced sympathoinhibition in rats. In many regions of the central nervous system, activation of cannabinoid-1 receptors presynaptically inhibits γ-aminobutyric acid (GABA) release, disinhibiting postsynaptic neurons. To determine if cannabinoid-1 receptor-mediated presynaptic inhibition of GABA release occurs in the NTS, we recorded miniature inhibitory postsynaptic currents in anatomically identified second-order baroreceptive NTS neurons in the presence of ionotropic glutamate receptor antagonists and tetrodotoxin. The cannabinoid-1 receptor agonists, WIN 55212-2 (0.3-30 μM) and methanandamide (3 μM) decreased the frequency of miniature inhibitory postsynaptic currents in a concentration-dependent manner, an effect that was blocked by the cannabinoid-1 receptor antagonist, N-(piperidin-1-yl)-5-(4-iodophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide (AM 251, 5 μM). Importantly, depolarization of second-order baroreceptive neurons decreased the frequency of miniature inhibitory postsynaptic currents; an effect which was blocked by the cannabinoid-1 receptor antagonist. The data indicate that depolarization of second-order baroreceptive NTS neurons induces endocannabinoid release from the neurons, leading to activation of presynaptic cannabinoid-1 receptors, inhibition of GABA release and subsequent enhanced baroreflex signaling in the NTS. The data suggest that endocannabinoid signaling in the NTS regulates short-term synaptic plasticity and provide a mechanism for endocannabinoid modulation of central baroreflex control., (Copyright © 2010 Elsevier B.V. All rights reserved.)

Chemoreflex afferent fibers terminate in the nucleus tractus solitarii (NTS), but the specific location of the NTS neurons excited by peripheral chemoreflex activation remains to be characterized. Here, the topographic distribution of chemoreflex sensitive cells at the commissural NTS was evaluated. To reach this goal, Fos-immunoreactive neurons (Fos-ir) were accounted in rostro-caudal levels of the intermediate and caudal commissural NTS, after intermittent chemoreflex activation with intravenous injection of potassium cyanide [KCN (80microg/kg) or saline (0.9%, vehicle), one injection every 3min during 30min]. In response to intermittent intravenous injections of KCN, a significant increase in the number of Fos-ir neurons was observed specifically in the lateral intermediate commissural NTS [(LI)NTS (82+/-9 vs. 174+/-16, cell number mean per section)] and lateral caudal commissural NTS [(LC)NTS (71+/-9 vs. 199+/-18, cell number mean per section)]. To evaluate the influence of baroreceptor-mediated inputs following the increase in blood pressure during intermittent chemoreflex activation, we performed an intermittent activation of the arterial baroreflex by intravenous injection of phenylephrine [1.5microg/kg iv (one injection every 3min during 30min)]. This procedure induced no change in Fos-ir in (LI)NTS (64+/-6 vs. 62+/-12, cell number mean per section) or (LC)NTS (56+/-15 vs. 77+/-12, cell number mean per section). These data support the involvement of the commissural NTS in the processing of peripheral chemoreflex, and provide a detailed characterization of the topographical distribution of activated neurons within this brain region.

In previous paper, we reported that stimulation of the nucleus raphe magnus (stim-NRM) inhibits the induction of retching by afferent vagal fibers (VAs). We performed the present study to identity the transmitter of inhibition and then the site. The following results were obtained in decerebrated and paralyzed dogs. 1) The induction of fictive retching was suppressed by i.v. injection of 5-HT, and by 4th ventricular administration of 5-HT or a 5-HT3-receptor (R) agonist, 1-(m-chlorophenyl)-biguanade hydrochloride (m-CPBG). 2) Both forms of suppression were antagonized by i.v. injection of ondansetron, a 5-HT3-R antagonist. 3) Administration of the antagonist into the 4th ventricle did not affect the induction or its suppression by stim-NRM. These results suggest that the transmission from VAs to neurons in the nucleus solitarius (NTS) is suppressed by 5-HT via 5-HT3-R. However, these results also suggest that both the transmitter and receptor are not involved in the induction of retching by VAs or in its suppression by the NRM. Next, we examined the site of suppression. Unitary firings of NTS neurons in response to pulse-train stimulation of VAs were not inhibited by NRM stimulation. Moreover, the firing of NTS neurons during the induction of retching by vagal stimulation did not significantly decrease with the superimposition of stim-NRM, although the induction of retching was completely suppressed. These results suggest that suppression of the induction of retching by the descending inhibitory system of pain did not occur in the synapse between afferent vagal fibers and NTS neurons. The site of suppression is discussed.

GABA receptors in the nucleus tractus solitarius (NTS) are known to play an important role in the mediation/modulation of various cardiovascular/respiratory functions. Vagal afferent activation of these neurones usually evokes an initial excitation followed by a long lasting inhibition. The present study examines the role of GABA(B) as well as GABA(A) receptors in the mediation of this inhibition in anaesthetized rats, using CGP 35348 and bicuculline, respective antagonists at these receptors, applied topically or by ionophoresis. Bicuculline delayed the onset and reduced the duration of this inhibition. The duration of this inhibition was further and significantly decreased when CGP 35348 was administered along with bicuculline but the delay in onset was unaffected. CGP 35348 application alone, had no effect on the vagal afferent-evoked inhibition. From intracellular recordings the early and late components of this inhibition were found to have reversal potentials close to E(Cl) and E(K), respectively. Therefore it is concluded that this inhibition is mediated by both GABA(A) and GABA(B) receptors, although GABA(B) mediated inhibition can only be unmasked when GABA(A) receptors are blocked.

Previously, we found that endocannabinoids acting at cannabinoid 1 receptors in the nucleus tractus solitarius prolonged baroreflex inhibition of renal sympathetic nerve activity in normotensive Sprague Dawley rats. The current study investigated whether endocannabinoid signaling was altered in spontaneously hypertensive rats, a model marked by elevated sympathetic activity and depressed baroreflex responses. The effects of endocannabinoids in the nucleus tractus solitarius on baroreflex control of renal sympathetic nerve activity evoked by systemic pressor changes or by direct stimulation of nucleus tractus solitarius neurons, which produced depressor and sympathoinhibitory responses, were studied in Sprague Dawley rats, Wistar Kyoto rats, and spontaneously hypertensive rats. Evoked responses were compared before and after microinjection of AM404, which prolonged actions of endogenous endocannabinoids, or microinjection of an endocannabinoid, anandamide, into the baroreceptive region of the nucleus tractus solitarius. AM404 microinjections significantly prolonged evoked sympathoinhibition in Sprague Dawley and Wistar Kyoto rats, but had little effect in spontaneously hypertensive rats. Microinjections of anandamide prolonged sympathoinhibition in Sprague Dawley rats, with lesser effects in Wistar Kyoto rats and no effects in spontaneously hypertensive rats. Parallel studies found that density of binding sites of endocannabinoids in the nucleus tractus solitarius was significantly reduced in spontaneously hypertensive rats versus the normotensive rats. Results indicate that attenuated function of the endocannabinoid system in the nucleus tractus solitarius of spontaneously hypertensive rats resulted in less modulation of baroreflex-evoked sympathoinhibition and that reduced cannabinoid 1 receptor density could contribute to blunted baroreflex-induced sympathoinhibition and elevated sympathetic tone characteristic of spontaneously hypertensive rats.

A consistent and prominent feature, observed across many species, including our neuromuscular blocked (NMB) rat preparation, is that obliterating the baroafferent inputs to the brainstem, e.g., by sinoaortic denervation (SAD), significantly increases blood pressure variability (BPV). The sources of the BPV, however, are not completely understood, but involve both the central and the peripheral mechanisms. The key central noise source is likely in the brainstem. Previously, in NMB rats, we showed that the maximum gain of the baroreflex system is in the very low frequency (VLF) range of 0.01-0.2 Hz. In this study, using the same NMB preparation, we demonstrated that, after SAD, there was a significant increase in the VLF power of the expiratory systolic blood pressure (EsBP) spectrum, but a decrease in the VLF power of the expiratory heart inter-beat-interval (EIBI) spectrum. Because dmNTS is the only major common anatomic node for the vascular sympathetic and the cardiac parasympathetic pathways, the opposite changes in the post-SAD VLF powers of the EsBP and EIBI spectra suggest that dmNTS is unlikely the major noise source for the post-SAD BPV. Supporting this finding, we found that the dmNTS evoked response to single pulse baroreflex afferent aortic depressor nerve (ADN) stimuli was substantially more reliable than the evoked systolic blood pressure responses to the same stimuli.

Located in the lower brainstem, the paratrigeminal nucleus (Pa5) is related to cardiorespiratory autonomic reflex functions. To characterize the structures' role in blood pressure regulation and baroreflex response, both resting cardiovascular parameters and reflex responses were evaluated during phenylephrine-produced pressor responses in non-anaesthetized rats with or without bilateral chemical Pa5 ablation. The Pa5-ablated animals, in contrast to the Pa5-intact control animals, presented increased resting arterial pressure (115+/-4 vs. 100+/-3 mm Hg), decreased heart (293+/-10 vs. 315+/-7 bpm) and increase of the respiratory (104+/-3 vs. 94+/-5 rpm) rates, larger pressor responses and reduced baroreflex index (1.6+/-0.2 vs. 2.8+/-0.2, p<0.05). The cardiovascular changes, compatible to those produced by nucleus of the solitary tract (NTS) lesions in non-anaesthetized rats, indicate a reduction of both the sympathetic and cardiac components of the baroreflex response. Further analyses showed the Pa5 mediates reflex responses to smaller blood pressure increases, while the NTS would be predominantly active in surges over 40 mm Hg. Thus, the integrity of the Pa5 is important for resting blood pressure maintenance as for a full baroreceptor response.

The lower esophageal sphincter (LES) and the crural diaphragm (CD) surrounding the esophagogastric junction are key components of the gastroesophageal reflex mechanism, which engages the vago-vagal brainstem circuitry. Although both components work in conjunction to prevent gastroesophageal reflux, little is known about the brain area(s) where this integration takes place. The aims of this study were to: (1) trace the brainstem circuitry associated with the CD and the LES, and (2) determine possible sites of convergence. Experiments were done in adult male ferrets. Under isoflurane anesthesia, recombinant strains of the transneuronal pseudorabies virus (PRV-151 or PRV-Bablu) or the monosynaptic retrograde tracer cholera toxin beta-subunit (CTb) were injected into either the CD or the LES. Following a survival period of 5-7 days, animals were euthanized, perfused and their brains removed for dual-labeling immunofluorescence processing. In animals injected with recombinants of PRV into the CD and the LES, distinct labeling was found in various brainstem nuclei including: area postrema, DMV, nucleus tractus solitarius (NTS), medial reticular formation (MRF) and nucleus ambiguous (NA). Double-labeled cells were only evident in the DMV, NTS and MRF. Injections of CTb into the CD or the LES resulted in retrograde labeling only in the DMV. These findings demonstrate the presence of a direct projection from the DMV to the CD. They further suggest that the neuronal connections responsible for CD or LES function are contained in circuitries that, though largely independent, may converge at the level of DMV, NTS and MRF.

The arterial baroreceptors detect changes in blood pressure and form the afferent limb of the baroreflex which acts to buffer changes in pressure through reciprocal regulation of the sympathetic and parasympathetic outflow. We have previously shown that the sympathetic and parasympathetic limbs of the baroreflex operate over different pressure ranges and hypothesised that these differences in regulation of heart rate and sympathetic activity could originate from the baroafferents. We tested this hypothesis using sequential baroafferent denervations in the decerebrate, arterially perfused rat preparation. We found that baroreflex control of heart rate is critically dependent upon the aortic arch afferents with relatively little contribution from the carotid sinuses. Indeed the baroreflex bradycardia was attenuated by 85% (n=7) when only one aortic depressor nerve was cut indicating a strongly synergistic interaction between aortic baroafferents. By contrast baroreflex sympathoinhibition was dependent on inputs from all four sites, and the stimulation of any single site could elicit robust sympathoinhibition. These findings were independent of the sequence of baroafferent nerve resection (n=15). Perfusion of the isolated carotid sinus (n=5) showed that it was possible to elicit baroreflex sympathoinhibition (and changes in vascular resistance) without any significant change in heart rate despite the use of strong stimuli (>100 mmHg) or repeated pulsatile stimuli. These results indicate fundamental differences in the responses elicited by stimulation of the afferents from the carotid and aortic barosensor sites and suggest that their actions within the nucleus of the solitary tract are functionally specified (sympathetic versus parasympathetic).

The carotid body receptors participate in glucose regulation sensing glucose levels in blood entering the cephalic circulation. The carotid body receptors information, is initially processed within the nucleus tractus solitarius (NTS) and elicits changes in circulating glucose and brain glucose uptake. Previous work has shown that gamma-aminobutyric acid (GABA) in NTS modulates respiratory reflexes, but the role of GABA within NTS in glucose regulation remains unknown. Here we show that GABA(B) receptor agonist (baclofen) or antagonists (phaclofen and CGP55845A) locally injected into NTS modified arterial glucose levels and brain glucose retention. Control injections outside NTS did not elicit these responses. In contrast, GABA(A) agonist and antagonist (muscimol or bicuculline) produced no significant changes in blood glucose levels. When these GABAergic drugs were applied before carotid body receptors stimulation, again, only GABA(B) agonist or antagonist significantly affected glycemic responses; baclofen microinjection significantly reduced the hyperglycemic response and brain glucose retention observed after carotid body receptors stimulation, while phaclofen produced the opposite effect, increasing significantly hyperglycemia and brain glucose retention. These results indicate that activation of GABA(B), but not GABA(A), receptors in the NTS modulates the glycemic responses after anoxic stimulation of the carotid body receptors, and suggest the presence of a tonic inhibitory mechanism in the NTS to avoid hyperglycemia.

The effects of emetic stimulation on the swallowing reflex were investigated in decerebrated rats. Hypoxia, gastric distension and LiCl administration were used as emetic stimulations. The swallowing reflex was elicited by electrical stimulation of the superior laryngeal nerve (SLN, 20 Hz, 3-5 V, 0.3 ms duration) for 20 s. To examine the effect of hypoxia, nitrogen gas was inhaled under artificial ventilation. There were significantly fewer swallows during a decrease in PO(2) than under air ventilation (p<0.05). The number of swallows during 3-ml stomach distension was significantly lower than that before distension (p<0.05). Intravenous administration of LiCl (100 mg/kg) also significantly reduced the number of swallows (p<0.05). The combination of SLN stimulation and emetic stimuli occasionally produced burst activity of abdominal muscles, which might be associated with the gag reflex. Both the gag and swallowing reflexes are well known to be mediated by the nucleus of the solitary tract. The physiological roles of the gag reflex and the swallowing reflex are considered to be reciprocal. Taken together, these results suggest that emetic stimulation inhibits the swallowing pattern generator via the nucleus of the solitary tract, which in turn facilitates the gag reflex.

To clarify the mechanism of site-specific effects of acupuncture on gastric motor function, we studied the simultaneous recording of gastric motility and electrocardiogram (ECG) for heart rate variability (HRV) analysis in conscious rats. Gastric motility and ECG were recorded before, during and after electroacupuncture (EA) at ST-36 (hind limb) or ST-25 (abdomen). EA at ST-36 significantly increased gastric motility and decreased the ratio of low frequency (LF)/high frequency (HF) of the HRV analysis. In contrast, EA at ST-25 significantly inhibited gastric motility and increased LF/HF ratio. There was a significant correlation observed between the changes of gastric motility and LF/HF ratio in response to EA. It is suggested that the stimulatory effect of EA at ST-36 on gastric motility is associated with its stimulatory effect on vagal activity. The inhibitory effect of EA at ST-25 on gastric motility is associated with its stimulatory effect on the sympathetic nerve activity.

Projections from the nucleus of tractus solitarius (NTS) to the caudal ventrolateral medulla (CVLM) are important in mediating autonomic reflexes. However, little is known about the cellular properties of the CVLM-projecting NTS neurons. In this study, the CVLM-projecting NTS neurons were retrogradely labeled by fluorescent microspheres injected into the CVLM. Whole cell voltage- and current-clamp recordings were performed on labeled NTS neurons in coronal brainstem slices. Compared with unlabeled neurons, the labeled NTS neurons had more depolarized resting membrane potentials, larger input resistance, and higher firing activity in response to depolarizing currents. Bath application of an ionotropic glutamate receptor antagonist kynurenic acid and a non-NMDA receptor antagonist CNQX significantly decreased the firing activity in the majority of labeled NTS neurons. In contrast, an NMDA receptor antagonist AP5 failed to alter the firing activity in labeled neurons tested. While the glycine receptor antagonist strychnine had no effect on the firing activity, blockade of GABA(A)receptors with bicuculline significantly increased the firing rate in the majority of labeled NTS neurons. Furthermore, CNQX blocked the majority of spontaneous excitatory postsynaptic currents (EPSCs) and evoked EPSCs elicited by stimulation of the tractus solitarius. The residual spontaneous and evoked EPSCs were abolished by the nicotinic receptor antagonist mecamylamine and the purinergic P2X receptor antagonist iso-PPADS. Finally, while bicuculline completely blocked the miniature inhibitory postsynaptic currents (IPSCs), the spontaneous and evoked IPSCs were abolished by a combination of bicuculline and strychnine in labeled NTS neurons. Collectively, these data suggest that the CVLM-projecting neurons are a population of neurons with distinctive membrane properties.

In the present study we evaluated the possible modulatory role of noradrenaline on the neurotransmission of the peripheral chemoreflex afferents in the caudal commissural NTS of awake rats. To reach this goal we performed a dose-response curve to microinjection of increasing dose of noradrenaline into the caudal commissural NTS of awake rats and then the threshold dose, which produces minor changes in the baseline mean arterial pressure, was selected to be used in the chemoreflex experiment. The peripheral chemoreflex was activated with KCN before and after bilateral microinjections of noradrenaline (5 nMol/50 nL, threshold dose) into the NTS. The data show that microinjection of noradrenaline into the caudal NTS produced a significant reduction in the pressor response to the chemoreflex 30 s after the injection when compared to the control response (30+/-6 vs. 49+/-3 mm Hg) but no significant changes in the bradycardic response. The data indicate that noradrenaline in the caudal commissural NTS of awake rats may play an important inhibitory neuromodulatory role on the processing of the pressor/sympathoexcitatory component of the chemoreflex.

Previous studies suggested that the following neuronal circuit participates in the induction of vomiting by afferent vagal stimulation in decerebrated paralyzed dogs: (1) afferent fibers of the vagus nerve, (2) neurons of the solitary nucleus (NTS), (3) neurons of the prodromal sign center near the semicompact part of the nucleus ambiguus (scAMB), (4) neurons of the central pattern generator in the reticular area adjacent to the compact part of nucleus ambiguus (cAMB), (5) respiratory premotor neurons in the caudal medulla, (6) motor neurons of the diaphragm and abdominal muscles. However, the commonality of this neuronal circuit in different species has not yet been clarified. Thus, this study was conducted to clarify this point. This study clarified for the first time that fictive vomiting in decerebrated paralyzed ferrets could be induced by vagal stimulation, and could be identified by centrifugal activity patterns of the phrenic and abdominal muscle nerves. The distributions of c-Fos immunoreactive neurons in the NTS, scAMB and cAMB areas in ferrets that exhibited fictive vomiting were denser than those in ferrets that did not. Application of the nonNMDA receptor antagonist into the 4th ventricle produced the reversible suppression of fictive vomiting. The NK1 receptor immunoreactive puncta were found in the reticular area adjacent to the scAMB. Microinjections of NK1 receptor antagonist into the reticular areas on both sides abolished fictive vomiting. All these results in the ferrets are identical with results previously obtained in dogs and cats. Therefore, this suggests that the above neuronal circuit commonly participates in the induction of emesis in these animal species.

This work aimed to study the respective effects of central alpha2-adrenergic receptors (alpha2-ARS) and I1 imidazoline receptors (I1Rs) in the facilitatory effects of imidazoline-like drugs on the reflex bradycardia (RB). Experiments were performed in anaesthetized rabbits. The reflex bradycardic response was induced by phenylephrine injected i.v. LNP 509, rilmenidine and dexmedetomidine were administered intracisternally (i.c.). LNP509 (1 mg/kg, i.c.), a ligand highly selective for I1Rs, induced hypotension (54+/-3 vs. 93+/-2 mm Hg) and bradycardia (260+/-13 vs. 322+/-13 beats/min) (p<0.05, n=5) but did not affect RB. Rilmenidine (1 microg/kg, i.c.), a hybrid ligand which binds to both I1 and alpha2-ARS, also decreased arterial pressure (61+/-2 vs. 101+/-2 mm Hg) and heart rate (260+/-4 vs. 308+/-8) (p<0.01, n=5); it potentiated the RB (maximum R-R interval: 284+/-17 vs. 196+/-6 ms) (p<0.05, n=5). Dexmedetomidine (1 microg/kg, i.c.), a ligand selective for alpha2-ARs, reduced blood pressure (53+/-3 vs. 104+/-2 mm Hg) and heart rate (246+/-4 vs. 312+/-8 beats/min) (p<0.05, n=5) and potentiated the RB (maximum R-R interval: 518+/-38 vs. 194+/-4 ms) (p<0.05, n=5). The potentiation of RB was much greater than that observed with rilmenidine and was significantly prevented by L-NNA injected centrally. This study shows that: (i) an exclusive action on I1Rs which decreases arterial pressure, does not potentiate the RB ii) activation of alpha2-ARs potentiates the RB (iii) the R-R prolongation caused by alpha2-ARs stimulation is prevented by central NOS inhibition.

The present study investigated the prenatal development of the cyto- and chemoarchitecture of the human nucleus of the solitary tract from 9 to 35 weeks, by using Nissl staining and immunoreactivity to calbindin, calretinin, tyrosine hydroxylase and GAP-43. The nucleus began to gain heterogeneity and show different subnuclei as early as 13 weeks, and approached cytoarchitectural maturation from 21 to 25 weeks. The subnuclear division pattern observed in the fetal nucleus of the solitary tract at 25 weeks was very similar to that of the adult. Neurons immunoreactive to calbindin first appeared in the medial gastrointestinal area of the nucleus at 13 weeks, particularly within a putative gelatinosus subnucleus, while calretinin immunoreactivity during fetal life suggested the possible presence of a central subnucleus. Tyrosine hydroxylase immunoreactive neurons were seen in the medial subdivisions of the nucleus of the solitary tract as early as 13 weeks, but the population continued to increase until 25 weeks. Strong GAP-43 immunoreactivity was also present in the nucleus of the solitary tract at 13 weeks, especially in the dorsolateral and commissural subnuclei, while at 21 weeks there was a significant decline of GAP-43 expression. Results from the chemoarchitectural study showed that the human nucleus of the solitary tract expressed various neurochemical substances at an early developmental age (13 weeks), even before cellular and neuropil maturation was fully attained. Expression of these factors may play an important role in establishment and integration of viscerosensory function in the nucleus.

Sensory information from the gastrointestinal (GI) tract is transmitted centrally via primary afferents that terminate within the nucleus of the tractus solitarius (NTS) and utilize glutamate as their major neurotransmitter. Neurons of the NTS integrate this sensory information and transmit it to parasympathetic preganglionic neurons of the dorsal motor nucleus of the vagus (DMV), as well as to other areas, using principally glutamate, GABA and norepinephrine as neurotransmitters. Although susceptible to modulation by a vast array of neurotransmitters, the glutamatergic NTS to DMV synapse seems to play a minor role in the tonic modulation of gastric vagal reflexes. GABAergic neurotransmission between the NTS and DMV, however, is of critical importance as its in vivo blockade induces dramatic effects on gastric tone, motility and secretion. In in vitro experiments, however, this synapse appears initially resistant to modulation by most exogenously applied neuromodulators. Using opioid peptides as a model, this review will discuss the remarkable plasticity of the NTS-DMV GABAergic synapse. Modulation of this synapse appears dependent upon the levels of cAMP within the brainstem circuit. In particular, this review will outline how vagal afferent inputs appear to dampen the cAMP-PKA system via tonic activation of metabotropic glutamate receptors. Removal of vagal sensory input, coincident activation of the cAMP-PKA system, or inhibition of group II metabotropic glutamate receptors, allows receptor trafficking to occur selectively at the level of the NTS-DMV GABAergic synapse. Thus, we propose that the state of activation of vagal sensory inputs determines the gastric motor response via selective engagement of GABAergic synapses. This mini-review is based upon a presentation given at the International Society for Autonomic Neuroscience meeting in Marseille, France in July 2005.

The arterial baroreceptor reflex is the major feedback control system that acts to stabilize blood pressure. Abnormalities of this reflex are considered to be an underlying mechanism in the cardiovascular diseases such as hypertension and heart failure. There is accumulating evidence, however, that central nervous system mechanisms are involved in the enhanced sympathetic drive that occurs in these disease states. This article reviews studies performed in our laboratory in which a gene transfer technique, in combination with other methods, was used to determine the functional role of the central control of cardiovascular regulation. We developed a technique to transfer adenovirus vectors encoding specific genes into the nucleus tractus solitarii (NTS) or the rostral ventral medulla (RVLM) of rats in vivo. We applied this technique to hypertensive rats as well as in mice with heart failure to explore the pathophysiological significance of nitric oxide, reactive oxygen species, and Rho-kinase.

In our previous studies, it was found that ketanserin enhanced baroreflex sensitivity (BRS) in rats and this effect was not blood pressure dependent. The present work was designed to investigate the effects of ketanserin on BRS within the nucleus tractus solitarus (NTS) and the rostral ventrolateral medulla (RVLM). In anesthetized rats, BRS were evaluated by the changes in depressor action of aortic nerve stimulation and bradycardiac response to rapid pressor action of intravenous phenylephrine. It was found that bilateral injection of ketanserin (250 pmol for each side) into the NTS not only significantly increased blood pressure, but also attenuated baroreflex function. Interestingly, when ketanserin was bilaterally injected into RVLM, a significant enhancement of BRS was found and there were no modifications in basal blood pressure and heart period. The present study demonstrates that ketanserin-induced enhancement of BRS mainly occurred within the RVLM, which might contribute to the systemic effects of ketanserin on BRS.

It is well known that central transmission of vago-vagal reflex within the nucleus of solitary tract (NST) plays an important role in the regulation of gastrointestinal functions. The present study was designed to assess the role of NST glutamate receptor mechanism in pancreatic secretion evoked by intraduodenal hypertonic saline (HS) in anesthetized rats. Intraduodenal infusion of HS significantly (P<0.01) stimulated pancreatic protein output (from 2.60+/-0.09 to 4.18+/-0.24 mg/15 min). Bilaterally microinjected L-glutamate (5 nmol) into the medial nucleus of solitary tract (mNST) produced a significant increase of pancreatic protein secretion (from 2.65+/-0.12 to 4.80+/-0.34 mg/15 min, P<0.01). Bilateral injection of glutamate receptor antagonist kynurenic acid (KYN, 5 nmol) into the mNST completely abolished the increase of pancreatic protein output stimulated by intraduodenal HS (from 4.28+/-0.21 to 2.83+/-0.19 mg/15 min). Either NMDA receptor antagonist dl-2-amino-5-phosphonopentanoic acid (AP5, 1.5 nmol) or AMPA/Kainate receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX, 1.5 nmol) injected into the mNST markedly attenuated (P<0.05) the pancreatic protein secretion stimulated by intraduodenal HS. In conclusion, these findings showed that blockade of the NST glutamate receptors, including NMDA and AMPA/Kainate receptors antagonized pancreatic secretion evoked by intraduodenal osmolality factor, and suggested that glutamate receptor mechanism within the NST contributed to the central regulation of pancreatic secretion.

The goal of this study was to investigate the sustained sympatho-excitation which occurs in sepsis and which accompanies the fall in blood pressure and to analyze its time-correlation with heart rate and the role of the baro-chemoreflexes. Rats anesthetized with pentobarbital were treated with lipolysaccharide (LPS) 20 mg/kg/20 min i.v. and mean blood pressure (MBP), heart rate (HR), rectal temperature and renal sympathetic nerve activity (RSNA) were recorded. LPS induced a fall in blood pressure, an increase in HR (+20%) and RSNA (+355%); the arterial PO2 and PCO2 remained stable and the injection was fatal within 4 h. Baroreceptor and chemoreceptor denervation accelerated the fall in MBP but did not change the survival time. Under those conditions; RSNA excitation was slightly more pronounced. During treatment with gallamine and under artificial respiration to avoid possible respiratory changes through the chemoreflex pathway, the effects of LPS remained, except for a decrease in arterial PO2. Electrolytic lesioning of the nucleus tractus solitarius or blocking the effects of baroreflex efferents by either an alpha1 or alpha2-adrenoceptor antagonists failed to alter the effects of LPS. After treatment with a beta-adrenoceptor antagonist, LPS increased RSNA but not HR and the survival time of the rats shortened. LPS administered i.c. (1 mg/kg) induced, with a short latency, effects comparable to those produced by i.v. injection. Surprisingly, the time correlation between RSNA and HR rhythms persisted when MBP dropped after LPS and moreover it reappeared in baroreceptor denervated rats after LPS. Thus under these conditions of altered baroreflex pathway and LPS induced sympathetic activation, the sympathetic output from the medulla appears to play a role in the correlation between heart rate and sympathetic nerve activity. These data indicate that the marked RSNA activation and the tachycardia are correlated and that the baroreflex and chemoreflex are not inhibited during sepsis but appear to be of minor importance in the sympathetic activation and in the blood pressure modifications.

Implantable gastric electric stimulation (GES) has been under investigation for the treatment of gastric motor disorders and obesity. However, possible central mechanisms involving the effects of GES on gastric function are unclear. The purpose of this study was to examine the effects of GES with different parameters on neuronal activity in the nucleus tractus solitarii (NTS) of the medulla. Extracellular potentials of single neurons in NTS were recorded in pentobarbital anesthetized, paralyzed, ventilated male rats. GES with four sets of parameters was applied for one minute: GES-A (6 mA, 0.3 ms, 40 Hz, 2 s-on and 3 s-off), GES-B (20 mA, 0.3 ms, 40 Hz, 2 s-on and 3 s-off), GES-C (6 mA, 6 ms, 40 Hz, 2 s-on and 3 s-off), and GES-D (6 mA, 200 ms, 12 imps/min). 35/118 (30%) neurons in NTS were responsive to gastric distension (GD, 20 mmHg, 20 s). Forty-one percent, 67%, 76% and 42% of all the responsive NTS neurons were affected by GES-A, -B, -C and -D, respectively. More NTS neurons with gastric inputs were affected with GES-C (19/25) than with GES-A (11/27, P<0.05) and GES-D (10/24, P<0.05). Maximal excitatory responses (17.9+/-2.6 imp/s) of NTS neurons to GES-C were significantly greater than GES-D (9.7+/-4.8 imp/s, P<0.05), whereas average duration of excitatory response (74.8+/-4.3 s) of NTS neurons to GES-B was significant longer than GES-A (60.3+/-3.3 s). Gastric electrical stimulation primarily has an excitatory effect on NTS neurons receiving input from the stomach; the central neuronal response to GES is enhanced with stimulation using an increased pulse width and/or amplitude. This modulatory effect of GES on the central neurons receiving vagal inputs may contribute to the neural mechanisms of GES therapy for the treatment of patients with obesity and gastric motility disorders.

We studied the role of benzodiazepine (BDZ) receptors in the commissural subnucleus of the nucleus tractus solitarius (commNTS) in chemoreceptor reflex in urethane-anesthetized, pancronium-immobilized, artificially ventilated and bilaterally vagotomized rats. A BDZ agonist, diazepam (1-4 micromol/kg), administered intravenously reduced resting phrenic nerve activity (PNA) and blood pressure (BP). Stimulation of carotid chemoreceptors induced an increase in PNA and an increase in BP. Diazepam inhibited this chemoreceptor reflex. The effects of intravenous injection of diazepam (4 micromol/kg) on the chemoreceptor reflex were antagonized by microinjection of the BDZ antagonist flumazenil (100 pmol) into the commNTS. Microinjection of flumazenil (100 pmol) alone had no effect on the basal PNA and BP, and the chemoreceptor reflex. These results suggest that BDZ receptors are present in the carotid chemoreceptor reflex pathway in the commNTS and potentiate GABA(A) transmission.

Previous studies have shown that microinjection of NMDA or non-NMDA receptor agonists into the nucleus tractus solitarius of anesthetized rats produces bradycardia and hypotension. In the present study, we evaluated the autonomic components of the cardiovascular responses to the microinjection of alpha-amine-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) into the nucleus tractus solitarius of awake rats. AMPA (0.05 nmol 50 nl(-1)) was microinjected into the nucleus tractus solitarius before and at 2, 10, 30 and 180 min after intravenous injection of methylatropine (2 mg kg(-1)). The control response to the microinjection of AMPA into the lateral aspect of the commissural nucleus tractus solitarius consisted of bradycardia and hypotension, (-190+/-20 bpm and -49+/-7 mm Hg), which were effectively blocked at 2 min (-5+/-2 bpm and -4+/-5 mm Hg), 10 min (-3+/-2 bpm and -6+/-3 mm Hg) and 30 min (-9+/-4 bpm and -5+/-5 mm Hg) after methylatropine. Although the bradycardic and hypotensive responses remained significantly reduced 180 min after injection of methylatropine, the responses tended to normalize. The data show that microinjection of AMPA into the rostral commissural nucleus tractus solitarius of awake rats produced intense bradycardia essentially mediated by parasympathetic excitation, which was the cause of the concomitant hypotensive response.

There is evidence suggesting that metabotropic receptors may play a role in the neurotransmission of the baroreflex in the nucleus tractus solitarius (NTS) of rats. In a recent study from our laboratory, we verified that microinjection of a metabotropic receptor agonist, trans-1-amino-1,3-cyclopentanediocarboxylic acid, into the NTS of awake and anesthetized rats produced baroreflex-like responses (hypotension and bradycardia). In the present study, we evaluated the possible role of L-glutamate metabotropic receptors of the NTS in the neuromodulation of the parasympathetic component of baroreflex activation in awake rats. Bilateral microinjection (50 nl) of a metabotropic receptor antagonist (alpha-methyl-4-carboxyphenylglycine, MCPG, 100 mM) into the rostral commissural NTS produced no change in the gain of the baroreflex bradycardia. In addition, microinjection of MCPG into the NTS produced no changes in baseline mean arterial pressure (MAP) or heart rate (HR), indicating that metabotropic receptors play no tonic role in the neurotransmission of the baroreflex. The dose of MCPG used to block the metabotropic receptors was effective in reducing the bradycardic and hypotensive responses to microinjection (50 nl) of trans-1-amino-1,3-cyclopentanediocarboxylic acid (5 mM) into the NTS. The data show that metabotropic glutamate receptors play no major role in the neuromodulation of the parasympathetic component of the baroreflex at the NTS level., (Copyright 2002 Elsevier Science B.V.)

Vago-vagal reflexes play an integral role in the regulation of gastrointestinal function. Although there have been a number of reports describing the effects of various stimuli on the firing rates of vagal afferent fibers and vagal motor neurons, little is known regarding the neurotransmitters that mediate the vago-vagal reflexes. In the present work, we investigated the role of glutamate in the vago-vagal reflex induced by gastrointestinal distention. Using single-cell recording techniques, we determined the effects of gastric and duodenal distention on the firing rates of gut-related neurons in the dorsal vagal complex, in the absence and presence of glutamate antagonists. Kynurenic acid, a competitive glutamate receptor antagonist, injected into the dorsal vagal complex, blocked the neuronal response of neurons in the dorsal motor nucleus of the vagus and the nucleus of the solitary tract to gastrointestinal distention. Injection of glutamate into the nucleus of the solitary tract produced inhibition of dorsal motor nucleus of the vagus neurons that were also inhibited by gastric and/or duodenal distention. Thus, the distention-induced inhibition of dorsal motor nucleus of the vagus neurons may be mediated by glutamate-induced excitation of gut-related nucleus of the solitary tract neurons. To investigate the role of the various glutamate receptor subtypes in the distention-induced events, we studied the effects of 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), a selective non-NMDA receptor antagonist, and DL-2-amino-5-phosphonopentanoic acid (DL-AP5), a selective NMDA receptor antagonist. CNQX injected into the dorsal vagal complex either blocked or attenuated the inhibitory response of the neurons in the dorsal motor nucleus of the vagus and nucleus of the solitary tract neurons to gastric and duodenal distention. In contrast, DL-AP5 had less effect, especially in the vago-vagal reflex elicited by gastric distention. The results suggest (1) distention activates vagal afferents in the gastrointestinal tract; (2) the central branches of the vagal afferents from the gut terminate in the nucleus of the solitary tract and release glutamate that mainly act on non-NMDA receptors; (3) glutamate activates the inhibitory neurons in the nucleus of the solitary tract that project to the dorsal motor nucleus of the vagus; and (4) the inhibitory neurotransmitter suppresses the activity of the dorsal motor nucleus of the vagus neurons. For the excitatory neuronal responses of the dorsal motor nucleus of the vagus neurons to gastrointestinal distention, the possible circuit is that the vagal afferents containing glutamate directly activate the receptors on the dendrites of the dorsal motor nucleus of the vagus., (Copyright 2002 Elsevier Science B.V.)

Apelin peptides are now known to be endogenous ligands at the orphan G-protein coupled receptor, APJ. Apelin and its receptor have been found in the brainstem and shown to have a role in haemodynamic homeostasis when injected intravenously. The physiological role of this peptide and its receptor centrally are yet to be elucidated. In this study, urethane anaesthetised, paralysed and ventilated male Sprague-Dawley rats (350-450 g, n=4) were used to investigate the action of apelin-13 microinjected directly into the nucleus tractus solitarius (NTS) and the rostral ventrolateral medulla (RVLM) on arterial pressure and phrenic nerve activity. Apelin-13 microinjections (4 mmol/l, 50 nl) into the NTS resulted in either apnea or decreased phrenic nerve discharge amplitude by up to 30%. In the RVLM, apelin-13 caused either a 100-200% increase, or a 10-30% increase in phrenic nerve discharge amplitude depending on the exact site of injection. Increases of 10-20 mm Hg in arterial pressure were also evoked from both the NTS and the RVLM. These data suggest a role for apelin-13 in arterial pressure and respiratory control in the NTS and the RVLM.

Previous studies have shown that catecholaminergic neurons in the nucleus tractus solitarius (NTS) with ascending projections to the subfornical organ (SFO) are highly sensitive to gamma-aminobutyric acid (GABA). To clarify the role of the GABAergic system in the NTS in the regulation of the activity of noradrenergic NTS projections to the SFO, the present study was carried out to investigate the effects of local administration (50 nl) of GABA, the GABA(A) agonist muscimol, the GABA(B) agonist baclofen, the GABA(A) antagonist bicuculline or the GABA(B) antagonist phaclofen into the NTS on the release of noradrenaline (NA) in the region of the SFO using microdialysis techniques in rats under urethane anesthesia. Microinjections of GABA (10(-4) - 10(-2) M) into the region of the NTS significantly decreased the NA release in the SFO area. Injections of either muscimol (10(-4) - 10(-2) M) or baclofen (10(-5) - 10(-3) M) into the NTS region significantly attenuated the NA release in the SFO area. Injections of bicuculline (10(-5) and 10(-4) M), but not phaclophen (10(-6) - 10(-4) M), into the NTS region significantly enhanced the NA release in the SFO area, suggesting that the GABAergic system in the NTS may tonically inhibit the NA release in the SFO area through a GABA(A) receptor mechanism. Neither injection of these drugs in any of the doses used in this study into the NTS region caused any significant changes in the NA release in the sites away from the SFO. Injections of vehicle (50 nl) into the NTS region had no significant effect on the NA release in either the SFO area or the sites away from the SFO. These results suggest that the GABAergic system in the NTS may serve to decrease the release of NA in the SFO area and the two types of GABA receptors are involved in the modulation of the NA release.

Synaptic terminals in the nucleus of the solitary tract (NTS) from axons originating in the central nucleus of the amygdala (CeA) are known to contain gamma-aminobutyric acid (GABA) immunoreactivity. Here, we have investigated whether such projections contain neuropeptides as putative co-transmitters. Somata in the medial and lateral CeA that were retrogradely labelled with cholera toxin B (CTb) injected into the commissural NTS were found to be immunoreactive for GABA, somatostatin (SOM), neurotensin (NT), vasoactive intestinal polypeptide (VIP) and nitric oxide synthase (NOS). Subpopulations of fibres in the NTS that were anterogradely labelled with biotin dextran amine (BDA) injected into the CeA and examined using both fluorescence and electron microscopy appeared to colocalise somatostatin, but not other neuropeptides. Their varicosities were observed in proximity to NTS neurones that were immunoreactive for the somatostatin receptor sst2A subtype, substance P (SP) NK1 receptor, and the GABAA receptor alpha3, beta1 and gamma2 subunits. This morphological evidence is consistent with the possibility of GABA-somatostatin co-transmission at synapses of some of the CeA projection neurones to NTS that might inhibit cardiovascular reflex responses in response to fear or emotion-related stimuli.

Previous studies have shown that the activation of 5-HT3 receptors in the nucleus tractus solitarii (NTS) increases the baseline mean arterial pressure (MAP). In the present study, we evaluated the possible involvement of NMDA receptors in this pressor response. Four days before the experiments, under tribromoethanol anesthesia, rats received two guide cannulas in the direction of the NTS, and 1 day before the experiments, under tribromoethanol anesthesia, the femoral artery was cannulated for pulsatile arterial pressure (PAP), MAP, and heart rate (HR) measurements. On the day of the experiments, 2-methyl-serotonin, a 5-HT3 agonist, was microinjected into the NTS after microinjection of saline or AP-5, a selective NMDA receptor antagonist. Microinjection of 2-methyl-serotonin (5 nmol/50 nl) into the NTS after the vehicle (saline) produced a significant increase in MAP (+ 20 +/- 5 mm Hg, n = 8) while microinjection of 2-methyl-serotonin after microinjection of AP-5 (10 nmol/50 nl) produced no change in baseline MAP (-1 +/- 3 mm Hg, n = 11). Microinjection of AP-5 into the NTS produced no significant changes in the baseline MAP and HR. The data show that the increase in MAP in response to microinjection of a 5-HT3 agonist into the NTS is dependent on NMDA receptors.

The pharmacology and anatomy of neurones in the nucleus tractus solitarius (NTS) have proved to be difficult to study in vivo because of their generally small size and high packing density. To overcome these problems, we have developed an approach that combines drug application through multibarrelled electrodes with juxtacellular labelling via an attached single-barrelled electrode followed by immunohistochemical processing. This approach has allowed us to assess the responses of individual NTS neurones in vivo to ionotropic glutamate receptor agonists and antagonists and then, to determine whether the neurones expressed the glutamate receptor subunits, GLUR2,3 and NMDAR2a,b. It should also be possible to extend these techniques further and correlate morphology with these features and to examine pharmacologically characterised, dye-filled neurones at the ultrastructural level.

We previously reported that intra-4th-ventricular (i.4th.v.) administration of a non-NMDA receptor antagonist, NBQX, abolished vagally induced retching. This study was undertaken to ascertain whether or not the neuronal response in the solitary tract nucleus (NTS) to vagal stimulation and the vago-vagal gastric reflexes induced by non-emetic stimulation are also abolished by NBQX with a similar latency as in the case of retching. Ketamine and thiopental- or chloralose-anesthetized dogs were decerebrated, and the dorsal surface of the medulla was exposed. This study consisted of two series of experiments. In the first series, extracellular neuronal responses in the NTS to pulse-train vagal stimulation were recorded. Effects of NBQX on the neural response and vagally induced fictive retching were observed. In the second series, effects of glutamate receptor antagonists on gastric corpus responses to esophageal or gastric antral distension were observed. Retching was abolished 5-15 min after an i.4th.v. application of NBQX. and the neuronal responses disappeared within 14 min after application in nine of 10 NTS neurons. On the other hand, corpus contractility was inhibited by esophageal distension, and inhibited and/or enhanced by antral distension. While the inhibitory responses disappeared within 17 min after NBQX, the enhanced response remained even after NBQX and vagotomy, but was abolished by i.v. administration of hexamethonium. These results suggest that adaptive relaxation in the corpus, as well as retching, may be mediated by glutaminergic vagal afferents and non-NMDA receptors in the NTS.

The modulation by 5-HT2 receptors in the nucleus tractus solitarius of the reflex bradycardia evoked by stimulation of peripheral baroreceptors and cardiopulmonary chemoreceptors, and their possible functional interactions with local NMDA receptors, were investigated in pentobarbital- and urethane-anaesthetized rats, respectively. Microinjection of the 5-HT2 receptor agonist, 2,5-dimethoxy-4-iodoamphetamine (0.1-0.5 pmol), into the nucleus tractus solitarius elicited a dose-dependent hypotension and bradycardia. Bilateral microinjections at the same site of a subthreshold dose of 2,5-dimethoxy-4-iodoamphetamine (0.05 pmol) significantly enhanced the aforementioned reflex-evoked bradycardiac responses. In contrast, local bilateral microinjections of the NMDA receptor antagonist, 2-amino-5-phosphonopentanoic acid (500 and 1000 pmol), reduced, in a dose-dependent manner, both reflex-evoked responses. The facilitatory effect of 2,5-dimethoxy-4-iodoamphetamine upon these reflex-evoked bradycardiac responses was prevented by prior local microinjection of low doses of either the selective 5-HT2 receptor antagonist, ketanserin (10 pmol), or 2-amino-5-phosphonopentanoic acid (100 pmol), which, on their own, did not affect the reflex-associated bradycardia. These data suggest that 5-HT2 receptors within the nucleus tractus solitarius participate in a facilitatory modulation of the reflex control of heart rate, probably through functional interactions with local NMDA receptors.

The activity of neurons in the region of the nucleus of the solitary tract (NTS) that were antidromically identified by electrical stimulation of the rat subfornical organ (SFO) was tested for a response to microiontophoretic application of gamma-aminobutyric acid (GABA), hemorrhage (10 ml/kg b.w.t.), or local administration of the chemical neurotoxin, 6-hydroxydopamine (6-OHDA), into the SFO stimulation site. Microiontophoretically (MIPh) applied GABA caused a decrease excitability in 22 out of 24 neurons tested, and the inhibition was blocked by MIPh-applied bicuculline, a GABAA antagonist, but not by phaclofen, a GABAB antagonist. Of these neurons that responded to GABA, 17 displayed an increase in neural firing in response to hemorrhage, while 5 were unresponsive. The occurrence of both antidromic spikes and post-stimulus inhibition of 9 out of 13 neurons tested was completely abolished by the injection of 6-OHDA into the SFO. These results suggest that neurons in the region of the NTS, which carry peripheral baroreceptor information to the SFO, receive GABAergic inhibitory inputs via a GABAA receptor mechanism, and imply that part of these neurons are catecholaminergic.

The importance of the integrity of the ipsilateral rostral ventrolateral medulla (RVLM) in the pressor response to activation of unilateral arterial chemoreceptors was evaluated. To achieve this goal, the left carotid body artery was ligated prior to the experiment and a guide cannula was implanted in the direction of the right RLVM, i.e. the side where the carotid body artery was kept intact. On the day of the experiment, the chemoreflex was activated with potassium cyanide (KCN, i.v.) before and after unilateral microinjection of kynurenic acid into the rostral or caudal aspect of the RVLM. The data indicated that microinjection of kynurenic into the rostral or caudal aspect of the RVLM produced no effect on the pressor response of chemoreflex activation. These data suggest that chemoreflex activation excites a neuronal network in which the processing of the sympatho-excitatory component of the chemoreflex is not restricted to an excitatory projection from the nucleus tractus solitarii to the ipsilateral RVLM.

Diabetic autonomic neuropathy is a common complication following prolonged diabetes. Alterations of cardiovascular reflexes contribute to the increased cardiovascular morbidity and mortality seen in diabetic patients. This study sought to better characterize these complications by investigating the afferent limb of the baroreceptor reflex in an experimental rat model of diabetes. Streptozotocin (STZ)-induced diabetic and euglycemic control rats were studied at 8- and 16-week time points after initiation of the experiment. Activation of the afferent limb of the baroreceptor reflex was assessed by measuring the numbers of c-Fos-immunoreactive (ir) neurons in the CNS site of termination of the baroreceptor afferent neurons, the nucleus of the solitary tract (NTS). Initial experiments established that baseline cardiovascular parameters and NTS expression of c-Fos-ir neurons were not different between diabetic and control rats at either time point. Phenylephrine (PE)-induced activation of baroreceptors resulted in a significant elevation in the numbers of c-Fos-ir neurons in the NTS of control rats. Although diabetic rats showed similar pressor responses to PE, the activation of c-Fos-ir neurons in the NTS of diabetic rats was significantly attenuated. At both 8 and 16 weeks, STZ-induced diabetic rats had significantly fewer c-Fos-ir neurons in the commissural NTS and in the caudal subpostrernal NTS when compared to the non-diabetic control animals receiving PE. These data suggest that STZ-induced diabetes, for a period of 8 and 16 weeks, results in reduced activity in the afferent baroreceptor input to the NTS, and are consistent with diabetes-induced damage to baroreceptor afferent nerves.

We compared the cardiorespiratory reflex responses evoked by noxious stimulation of the forelimb and cornea. Due to the depressant effects of anaesthesia on visceral reflexes we compared data from an unanaesthetised decerebrate rat model--the working heart-brainstem preparation (WHBP), with the anaesthetised rat. In both experimental models stimulation of the forelimb (mechanical pinch) evoked a tachycardia (WHBP: 19 +/- 2 bpm) and a decrease in respiratory cycle length (WHBP: from 4.1 +/- 0.2 to 2.3 +/- 0.1 s). The magnitude of response in anaesthetised animals depended on anaesthetic depth. Mechanical stimulation of the cornea evoked a bradycardia (-49.2 +/- 4.8 bpm) and an increase in respiratory cycle length from 4 +/- 0.36 to 5.88 +/- 0.2 s which was only present in the WHBP. In the WHBP activation of forelimb and corneal nociceptors both elicited significant pressor effects; in anaesthetised rats there were inconsistent changes in arterial pressure. To determine a role for the nucleus of the solitary tract (NTS) in mediating nociceptive evoked responses in the WHBP, synaptic transmission was blocked reversibly following bilateral microinjections of cobalt chloride. The heart rate responses evoked from either forelimb or corneal nociceptors were attenuated by approximately 50% (P < 0.05). A similar effect was observed using isoguvacine, a GABAA receptor agonist, to hyperpolarise NTS neurones. In conclusion, activation of forelimb and corneal nociceptors evoked contrasting patterns of cardiorespiratory response in the WHBP while in the anaesthetised rat the magnitude of the cardiorespiratory response to forelimb stimulation was quantitatively dependent on anaesthetic dose. In the WHBP, NTS neurones appear important for mediating the cardiac component of the reflex response following stimulation of nociceptive reflex pathways.

In the present study we evaluated the effects of bilateral microinjection of muscimol (a GABA(A) receptor agonist) and baclofen (a GABA(B) receptor agonist) into the lateral commissural nucleus tractus solitarii (NTS) of awake rats on the gain of the baroreflex (BG) activated by a short duration (10-15 s) infusion of phenylephrine (Phe, 2.5 microg/0.05 ml, i.v.). Microinjection of muscimol (50 pmol/50 nl, n=8) into the NTS produced a significant increase in baseline mean arterial pressure ((MAP) 122+/-6 vs. 101+/-2 mmHg), no changes in baseline heart rate (HR) and a reduction in BG (-1.59+/-0. 1 vs. -0.69+/-0.1 beats/mmHg). Microinjection of baclofen (6.25 pmol/50 nl, n=6) into the NTS also produced a significant increase in baseline MAP (138+/-5 vs. 103+/-2 mmHg), no changes in baseline HR and a reduction in BG (-1.54+/-0.3 vs. -0.53+/-0.2 beats/mmHg). Considering that the reduction in BG could be secondary to the increase in MAP in response to microinjection of muscimol (n=6) or baclofen (n=7) into the NTS, in these two groups of rats we brought the MAP back to baseline by infusion of sodium nitroprusside (NP, 3.0 microg/0.05 ml, i.v.). Under these conditions, we verified that the BG remained significantly reduced after muscimol (-1.49+/-0.2 vs. -0.35+/-0.2 beats/mmHg) and after baclofen (-1.72+/-0.2 vs. -0.33+/-0.2 beats/mmHg) when compared to control. Reflex tachycardia was observed during the normalization of MAP by NP infusion and, in order to prevent the autonomic imbalance from affecting BG, we used another group of rats treated with atenolol (5 mg/kg, i.v.), a beta1 receptor antagonist. In rats previously treated with atenolol and submitted to NP infusion, we verified that BG remained reduced after microinjection of muscimol or baclofen into the NTS. The data show that activation of GABA(A) and GABA(B) receptors, independently of the changes in the baseline MAP or HR, inhibited the neurons of the NTS involved in the parasympathetic component of the baroreflex.

Serotonin has been implicated as having a modulatory action on NTS neurones mediating vagal inputs controlling the cardiorespiratory system. Since raphe pallidus and raphe obscurus both send serotonergic projections to the NTS, we have investigated a neuromodulatory role for these structures on the cardiopulmonary reflex. A multibarrelled microelectrode positioned around the level of the area postrema was placed at varying depths into mid-line brainstem structures and the effect of glutamate stimulation on the cardiopulmonary reflex tested. Excitatory chemical stimulation in the region of raphe pallidus, but not raphe obscurus, attenuated significantly the respiratory and bradycardic components of the cardiopulmonary reflex. This attenuation was reversed by an NTS microinjection of RS-39604, a selective 5-HT, receptor antagonist. We propose that neurones in raphe pallidus that project to the NTS can release serotonin which acts via 5-HT4 receptors to attenuate the reflex phrenic nerve activity and heart rate components of the cardiopulmonary reflex.