Splanchnic Sympathetic Nerve Discharge Research Articles

Blockade of central angiotensin II AT1 receptors attenuates acute intermittent hypoxia‐induced sympathetic long‐term facilitation (S‐LTF)

After exposure to acute intermittent hypoxia (AIH) and while undergoing continuous normoxic ventilation, a progressive increase of sympathetic nerve discharge (SND) is observed in the anesthetized rat. This phenomenon, referred to as post‐AIH sympathetic long‐term facilitation (S‐LTF), involves neuroplasticity resulting, at least in part, from repetitive arterial chemoreceptor activation, but the underlying mechanisms remain to be determined. Evidence indicates that post‐AIH S‐LTF is largely unaffected by intravenous injection of the angiotensin II (AngII) AT1 receptor antagonist losartan. Because AIH repetitively increases renal SNA in associated with large amplitude reductions of arterial pressure and thus renal perfusion pressure, both of which potently stimulate the renin‐AngII system, we hypothesized that central AngII actions contribute to post‐AIH S‐LTF. Anesthetized male rats received an injection (2 μl) of losartan (40 pmol) or vehicle (aCSF) into a lateral cerebral ventricle 20 minutes before exposure to a 1 hour AIH protocol, which consisted of 10 cycles of changing FiO2 from 100% to 15% every 3 minutes. Losartan significantly attenuated (p<0.001) the increase of splanchnic SND caused by AIH (vehicle: 112±34%, n=4 vs losartan: 27±7%, n=7). These data indicate that AngII acting at central AT1 receptors contributes to post‐AIH S‐LTF. When considered together with literature evidence, we conclude that the AngII signal of AIH is generated centrally, not peripherally.Support or Funding InformationNIH HL088052 & AHA 25710176 (GMT)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Effects of acute and chronic systemic methamphetamine on respiratory, cardiovascular and metabolic function, and cardiorespiratory reflexes.

Methamphetamine (METH) abuse is escalating worldwide, with the most common cause of death resulting from cardiovascular failure and hyperthermia; however, the underlying physiological mechanisms are poorly understood. Systemic administration of METH in anaesthetised rats reduced the effectiveness of some protective cardiorespiratory reflexes, increased central respiratory activity independently of metabolic function, and increased heart rate, metabolism and respiration in a pattern indicating that non-shivering thermogenesis contributes to the well-described hyperthermia. In animals that showed METH-induced behavioural sensitisation following chronic METH treatment, no changes were evident in baseline cardiovascular, respiratory and metabolic measures and the METH-evoked effects in these parameters were similar to those seen in saline-treated or drug naïve animals. Physiological effects evoked by METH were retained but were neither facilitated nor depressed following chronic treatment with METH. These data highlight and identify potential mechanisms for targeted intervention in patients vulnerable to METH overdose. Methamphetamine (METH) is known to promote cardiovascular failure or life-threatening hyperthermia; however, there is still limited understanding of the mechanisms responsible for evoking the physiological changes. In this study, we systematically determined the effects on both autonomic and respiratory outflows, as well as reflex function, following acute and repeated administration of METH, which enhances behavioural responses. Arterial pressure, heart rate, phrenic nerve discharge amplitude and frequency, lumbar and splanchnic sympathetic nerve discharge, interscapular brown adipose tissue and core temperatures, and expired CO2 were measured in urethane-anaesthetised male Sprague-Dawley rats. Novel findings include potent increases in central inspiratory drive and frequency that are not dependent on METH-evoked increases in expired CO2 levels. Increases in non-shivering thermogenesis correlate with well-described increases in body temperature and heart rate. Unexpectedly, METH evoked minor effects on both sympathetic outflows and mean arterial pressure. METH modified cardiorespiratory reflex function in response to hypoxia, hypercapnia and baroreceptor unloading. Chronically METH-treated rats failed to exhibit changes in baseline sympathetic, cardiovascular, respiratory and metabolic parameters. The tonic and reflex cardiovascular, respiratory and metabolic responses to METH challenge were similar to those seen in saline-treated and drug naive animals. Overall, these findings describe independent and compound associations between physiological systems evoked by METH and serve to highlight that a single dose of METH can significantly impact basic homeostatic systems and protective functions. These effects of METH persist even following chronic METH treatment.

Respiratory, metabolic and cardiac functions are altered by disinhibition of subregions of the medial prefrontal cortex

The prefrontal cortex (PFC) is referred to as the visceral motor cortex; however, little is known about whether this region influences respiratory or metabolic outflows. The aim of this study was to describe simultaneous changes in respiratory, metabolic and cardiovascular functions evoked by disinhibition of the medial PFC (mPFC) and adjacent lateral septal nucleus (LSN). In urethane-anaesthetized rats, bicuculline methiodide was microinjected (2 mm; GABA-A receptor antagonist) into 90 sites in the mPFC at 0.72-4.00 mm from bregma. Phrenic nerve amplitude and frequency, arterial pressure, heart rate, splanchnic and lumbar sympathetic nerve activities (SNA), expired CO2, and core and brown adipose tissue temperatures were measured. Novel findings included disturbances to respiratory rhythm evoked from all subregions of the mPFC. Injections into the cingulate cortex evoked reductions in central respiratory function exclusively, whereas in ventral sites, particularly the infralimbic region, increases in respiratory drive and frequency, and metabolic and cardiac outflows were evoked. Disinhibition of sites in surrounding regions revealed that the LSN could evoke cardiovascular changes accompanied by distinct oscillations in SNA, as well as increases in respiratory amplitude. We show that activation of neurons within the mPFC and LSN influence respiratory, metabolic and cardiac outflows in a site-dependent manner. This study has implications with respect to the altered PFC neuronal activity seen in stress-related and mental health disorders, and suggests how basic physiological systems may be affected.

Glutamatergic activities in neonatal rat spinal cord heterogeneously regulate single-fiber splanchnic nerve discharge

Kynurenic acid (KYN) is a metabolite of tryptophan and is involved in various neurological disorders. Using whole-bundle nerve recording techniques, we previously observed that applications of KYN to block endogenous ionotropic glutamate receptor activities in neonatal rat spinal cords in vitro cause a reversible fluctuation of splanchnic sympathetic nerve discharge (SND). We hypothesized that the SND fluctuation was due to a heterogeneous single-fiber response. To detail individual fiber activities, we used the so-called 'oligofiber recordings'. Spontaneous single-fiber activities were recorded from the collagenase-dissociated splanchnic nerve fascicles. Applications of KYN increased, decreased or did not change firing rates. The heterogeneous responses in spontaneous spiking activities were confirmed by applications of APV or CNQX, suggesting an effect mediated by endogenous NMDA- or non-NMDA receptor activities. In addition to changes in firing rates, apparent drug-induced changes in firing patterns were also observed in some fiber activities. Using the oligofiber recording techniques, we confirmed a differential role of endogenous ionotropic glutamate receptor activities in regulating sympathetic outflows from the spinal cord of neonatal rats. Fine-tuning of ionotropic glutamate receptor activities in the spinal cord may serve as a simple way for heterogeneous regulation of various sympathetic-targeting tissues.

Chemosensory control by commissural nucleus of the solitary tract in rats

The commissural nucleus of the solitary tract (commNTS) is a main area that receives afferent signals involved in the cardiovascular and respiratory control like those related to chemoreceptor activation, however, the importance of the commNTS for the cardiorespiratory responses to chemoreceptor activation is still controversial. In the present study, we investigated the cardiorespiratory responses to hypoxia or hypercapnia in anesthetized and conscious rats treated with injections of the GABA-A agonist muscimol into the caudal portion of the commNTS. Male Holtzman rats (280-300 g) were used. In conscious rats that had a stainless steel cannula previously implanted into the commNTS, the injection of muscimol (2 mM) into the commNTS reduced the pressor response (16±2 mmHg, vs. saline: 36±3 mmHg) and the increase in ventilation (250±17 ml/min/kg, vs. saline: 641±28 ml/min/kg) produced by hypoxia (8-10% O(2)). In urethane anesthetized rats, the injection of muscimol into the commNTS eliminated the pressor response (5±2 mmHg, vs. saline: 26±5 mmHg) and the increase in phrenic nerve discharge (PND) (20±6%, vs. saline: 149±15%) and reduced the increase in splanchnic sympathetic nerve discharge (sSND) (93±15%, vs. saline: 283±19% of baseline) produced by hypoxia. However, muscimol injected into the commNTS did not change hypercapnia (8-10% CO(2)) induced pressor response or the increase in the sSND or PND in urethane anesthetized rats or the increase in ventilation in conscious rats. The present results suggest that the cardiorespiratory responses to hypoxia are strongly dependent on the caudal portion of the commNTS, however, this area is not involved in the responses to hypercapnia.

Contribution of excitatory amino acid receptors of the retrotrapezoid nucleus to the sympathetic chemoreflex in rats

In the present study, we evaluated the role of glutamatergic mechanisms in the retrotrapezoid nucleus (RTN) in changes of splanchnic sympathetic nerve discharge (sSND) and phrenic nerve discharge (PND) elicited by central and peripheral chemoreceptor activation. Mean arterial pressure (MAP), sSND and PND were recorded in urethane-anaesthetized, vagotomized, sino-aortic denervated and artificially ventilated male Wistar rats. Hypercapnia (10% CO(2)) increased MAP by 32 ± 4 mmHg, sSND by 104 ± 4% and PND amplitude by 101 ± 5%. Responses to hypercapnia were reduced after bilateral injection of the NMDA receptor antagonist d,l-2-amino-5-phosphonovalerate (AP-5; 100 mm in 50 nl) in the RTN (MAP increased by 16 ± 3 mmHg, sSND by 82 ± 3% and PND amplitude by 63 ± 7%). Bilateral injection of the non-NMDA receptor antagonist 6,7-dinitro-quinoxaline-2,3-dione (DNQX; 100 mm in 50 nl) and the metabotropic receptor antagonist (+/-)-α-methyl-4-carboxyphenylglycine (MCPG; 100 mm in 50 nl) in the RTN did not affect sympathoexcitatory responses induced by hypercapnia. Injection of DNQX reduced hypercapnia-induced phrenic activation, whereas MCPG did not. In animals with intact carotid chemoreceptors, bilateral injections of AP-5 and DNQX in the RTN reduced increases in MAP, sSND and PND amplitude produced by intravenous injection of NaCN (50 μg kg(-1)). Injection of MCPG in the RTN did not change responses produced by NaCN. These data indicate that RTN ionotropic glutamatergic receptors are involved in the sympathetic and respiratory responses produced by central and peripheral chemoreceptor activation.

High-fat diet is associated with blunted splanchnic sympathoinhibitory responses to gastric leptin and cholecystokinin: implications for circulatory control

Gastric leptin and cholecystokinin (CCK) act on vagal afferents to induce cardiovascular effects and reflex inhibition of splanchnic sympathetic nerve discharge (SSND) and may act cooperatively in these responses. We sought to determine whether these effects are altered in animals that developed obesity in response to a medium high-fat diet (MHFD). Male Sprague-Dawley rats were placed on a low-fat diet (LFD; n = 8) or a MHFD (n = 24) for 13 wk, after which the animals were anesthetized and artificially ventilated. Arterial pressure was monitored and blood was collected for the determination of plasma leptin and CCK. SSND responses to leptin (15 μg/kg) and CCK (2 μg/kg) administered close to the coeliac artery were evaluated. Collectively, MHFD animals had significantly higher plasma leptin but lower plasma CCK levels than LFD rats (P < 0.05), and this corresponded to attenuated or reversed SSND responses to CCK (LFD, -21 ± 2%; and MHFD, -12 ± 2%; P < 0.05) and leptin (LFD, -6 ± 2%; and MHFD, 4 ± 1%; P < 0.001). Alternatively, animals on the MHFD were stratified into obesity-prone (OP; n = 8) or obesity-resistant (OR; n = 8) groups according to their weight gain falling within the upper or lower tertile, respectively. OP rats had significantly higher resting arterial pressure, adiposity, and plasma leptin but lower plasma CCK compared with LFD rats (P < 0.05). The SSND responses to CCK or leptin were not significantly different between OP and OR animals. These results demonstrate that a high-fat diet is associated with blunted splanchnic sympathoinhibitory responses to gastric leptin and CCK and may impact on sympathetic vasomotor mechanisms involved in circulatory control.

Inhibition of the caudal pressor area reduces cardiorespiratory chemoreflex responses

The caudal pressor area (CPA) is a brainstem area located close to the spinal cord. The activation of the CPA increases sympathetic activity and mean arterial pressure (MAP) by mechanisms dependent on the commissural nucleus of the solitary tract (commNTS) and rostroventrolateral medulla, however, the signals that activate the CPA to produce these responses are still unknown. Therefore, in the present study, we investigated the activity of glutamatergic and GABAergic mechanisms from the CPA and commNTS in rats exposed to hypoxia and the effects of the inhibition of CPA neurons on cardiorespiratory responses to peripheral chemoreceptor activation with i.v. sodium cyanide (NaCN). Male Sprague–Dawley rats (250–280 g, n=5–8/group) were used. In conscious rats, most of the commNTS neurons (66±11%) and part of the CPA neurons (36±7%) activated by hypoxia (8% O2) were glutamatergic (contained VGLUT2mRNA). Small part of the neurons activated during hypoxia was GABAergic (contained GAD-67mRNA) in the commNTS (9±4%) or the CPA (6±2%). In urethane anesthetized rats, the inhibition of CPA neurons with bilateral injections of muscimol (GABA-A agonist, 2 mM) reduced baseline MAP, splanchnic sympathetic nerve discharge (SND) and phrenic nerve discharge (PND). Muscimol into the CPA also reduced by around 50% the pressor and sympathoexcitatory responses and the increase in PND to peripheral chemoreceptor activation with NaCN (50 μg/kg i.v.), without changing sympathetic baroreflex responses. These data suggest that CPA mechanisms facilitate cardiorespiratory responses to peripheral chemoreflex activation. Immunohistochemistry results also suggest that at least part of the CPA mechanisms activated by hypoxia is glutamatergic.

Ventrolateral medulla mechanisms involved in cardiorespiratory responses to central chemoreceptor activation in rats

A rise in arterial Pco(2) stimulates breathing and sympathetic activity to the heart and blood vessels. In the present study, we investigated the involvement of the retrotrapezoid nucleus (RTN) and glutamatergic mechanisms in the Bötzinger/C1 region (Bötz/C1) in these responses. Splanchnic sympathetic nerve discharge (sSND) and phrenic nerve discharge (PND) were recorded in urethane-anesthetized, sino-aortic-denervated, vagotomized, and artificially ventilated rats subjected to hypercapnia (end-expiratory CO(2) from 5% to 10%). Phrenic activity was absent at end-expiratory CO(2) of 4%, and strongly increased when end-expiratory CO(2) reached 10%. Hypercapnia also increased sSND by 103 ± 7%. Bilateral injections of the GABA-A agonist muscimol (2 mM) into the RTN eliminated the PND and blunted the sSND activation (Δ = +56 ± 8%) elicited by hypercapnia. Injections of NMDA receptor antagonist AP-5 (100 mM), non-NMDA receptor antagonist 6,7-dinitro-quinoxaline-2,3-dione (DNQX; 100 mM) or metabotropic glutamate receptor antagonist (+/-)-alpha-methyl-4-carboxyphenylglycine (MCPG; 100 mM) bilaterally into the Bötz/C1 reduced PND (Δ = +43 ± 7%, +52 ± 6% or +56 ± 11%, respectively). MCPG also reduced sSND (Δ = +41 ± 7%), whereas AP-5 and DNQX had no effect. In conclusion, the increase in sSND caused by hypercapnia depends on increased activity of the RTN and on metabotropic receptors in the Bötz/C1, whereas PND depends on increased RTN activity and both ionotropic and metabotropic receptors in the Bötz/C1.

Important GABAergic mechanism within the NTS and the control of sympathetic baroreflex in SHR

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 (25 pmol/50 nl) or the GABA-B antagonist CGP 35348 (5 nmol/50 nl). Baseline MAP in SHR was higher than the WKY rats (SHR: 153 ± 5, vs. WKY: 112 ± 6 mm Hg, p < 0.05). Bilateral injection of bicuculline or CGP 35348 into the NTS induced a transient (5 min) reduction in MAP (∆ = −26 ± 4 and −41 ± 6 mm Hg, respectively vs. saline ∆ = + 4 ± 3 mm Hg, 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.

Supraspinal contribution to splanchnic sympathetic activity in neonatal mouse and rat brainstem–spinal cord in vitro

Under optimal in vitro conditions, isolated spinal cords of neonatal Sprague–Dawley (SD) rats spontaneously generate sympathetic nerve discharge (SND). To aid future studies involving genetically modified mice, we established a preparation to assess SND generation within the mouse spinal cord. Brainstem–spinal cord–splanchnic nerve preparations of neonatal 129S6/SvEvTac (129S6) mice, C57BL/6 (B6) mice, Long-Evan (LE) rats, and SD rats were used. The contributions of the brainstem to splanchnic SND (sSND) were not significantly affected by cervical cord transections in LE and SD rats. However, the transections caused approximately a 70% reduction in sSND in both mice species. Power spectral analyses characterized distinct features of sSND oscillations. With intact brainstem–spinal cord, comparable maximal power peaks at ∼ 1–2 Hz were observed in mouse and rat spectra, although the spectral peak widths were broader in mice. Cervical cord transections reduced the maximal peak powers and the peak widths in mice but not in rats. For comparisons across animal groups, the amounts of sSND were normalized to ambient current noise and expressed in signal/noise units. Similar amounts of normalized sSND were recorded from mice and rats with intact brainstem–spinal cords. However, the level of mouse sSND was reduced following cervical cord transection, whereas rat sSND was not. Our results demonstrate a species related difference in sSND recorded from neonatal rat and mouse spinal cord preparations in vitro. The current experimental model is applicable to evaluate the SND strength in neonatal rodents of various genetic backgrounds.

HIGH FAT DIET ALTERS SPLANCHNIC SYMPATHOINHIBITORY RESPONSES TO GASTRIC HORMONES

Gastric leptin and cholecystokinin (CCK) act on vagal afferents to induce reflex inhibition of splanchnic sympathetic nerve discharge (SSND), and may act co‐operatively in this response. We sought to determine whether this response is altered in animals exposed to a medium high fat diet (MHFD). Male Sprague‐Dawley rats were placed on a low‐fat diet (LFD; n=8) or a MHFD (n=24) for 13 weeks, after which animals were anesthetised and artificially ventilated. Baseline arterial pressure (AP) was measured and blood was collected for determination of plasma leptin and CCK. SSND responses to leptin (15 μg/kg) and CCK (2 μg/kg) administered close to the celiac artery were evaluated. Animals on the MHFD were grouped as obesity prone (OP; n=8) or obesity resistant (OR; n=8); the effects of diet alone were also examined. OP rats had significantly higher AP, fat pad mass and plasma leptin but lower plasma CCK, compared to LFD rats (P &lt; 0.05 for all). Collectively, MHFD animals had elevated leptin but lower CCK levels than LFD rats (P &lt; 0.05 for both) and this corresponded with attenuated/reversed SSND responses to CCK (LFD: −21 ± 2 Units vs MHFD −10 ± 2 Units; P &lt; 0.05) and leptin (LFD: −6 ± 2 Units vs MHFD: 4 ± 1 Units; P &lt; 0.001). High fat diets may induce leptin resistance that reduces the sensitivity of vagal afferents to gastrointestinal hormones. Increased gastrointestinal vascular resistance may contribute to hypertension in obesity.

Gastric leptin: a novel role in cardiovascular regulation

Gastric-derived leptin affects satiety and gastrointestinal function via vagal mechanisms and has been shown to interact with the gut hormone cholecystokinin (CCK). CCK selectively inhibits splanchnic sympathetic nerve discharge (SND) and the activity of a subset of presympathetic vasomotor neurons in the rostroventrolateral medulla (RVLM). The present study sought to examine the effects of gastric leptin on arterial pressure (AP), heart rate (HR), SND, and RVLM neuronal activity to determine whether its effects on cardiovascular regulation are dependent on CCK(1) receptors and vagal afferent transmission. To mimic gastric leptin, leptin (15-30 microg/kg) was administered close to the coeliac artery in anesthetized, artificially ventilated Sprague-Dawley rats. Within 5 min, leptin selectively decreased the activity of RVLM neurons also inhibited by CCK (-27 +/- 4%; P < 0.001; n = 15); these inhibitory effects were abolished following administration of the CCK(1) receptor antagonist lorglumide. Leptin significantly decreased AP and HR (-10 +/- 2 mmHg, P < 0.001; and -8 +/- 2 beats/min, P < 0.01; n = 35) compared with saline (-1 +/- 2 mmHg, 3 +/- 2 beats/min; n = 30). In separate experiments, leptin inhibited splanchnic SND compared with saline (-9 +/- 2% vs. 2 +/- 3%, P < 0.01; n = 8). Bilateral cervical vagotomy abolished the sympathoinhibitory, hypotensive, and bradycardic effects of leptin (P < 0.05; n = 6). Our results suggest that gastric leptin may exert acute sympathoinhibitory and cardiovascular effects via vagal transmission and CCK(1) receptor activation and may play a separate role to adipose leptin in short-term cardiovascular regulation.

Role of GABAergic receptors within the nucleus of the solitary tract in spontaneously hypertensive rats

Inhibitory neurotransmission plays a crucial role in the processing of sensory afferent signals in the nucleus of the solitary tract (NTS). Gamma‐aminobutyric acid (GABA) neurons are scattered throughout NTS. Previous studies demonstrated that alterations in GABA mediated neural transmission in the NTS contribute to the attenuation of the baroreceptor reflex observed in SHR. Here, we test the hypothesis that enhanced stimulation of GABA‐A and B receptors in the NTS in SHR are responsible for the attenuation of the sympathetic baroreflex, but not the chemoreflex. Under urethane/chloralose anesthesia, baseline arterial pressure (AP) in SHR was higher than the Wistar Kyoto (WKY) rats (SHR: 146 ± 5, vs. WKY: 112 ± 6 mmHg). The effects of baroreflex and chemoreflex on AP, splanchnic sympathetic nerve discharge (sSND) and phrenic nerve discharge (PND) were test before and after bilateral injection of the GABA‐A antagonist (bicuculline) or the GABA‐B antagonist (CGP 35348) within the NTS. The baroreflex range and gain were increased after bicuculline and CGP 35348 injection into the NTS, but had no effect on sympathoexcitatory and increase PND produced by chemoreflex activation. Bilateral injection of bicuculline and GCP 35348 within the NTS in SHR produced a pronounced decrease in AP compared with WKY (Δ = ‐15 ± 4 and ‐33 ± 6 mmHg, respectively vs. WKY: Δ = ‐5 ± 4 mmHg), but did not change PND. These results suggest that alterations in GABA‐mediated neural transmission in the NTS contribute to the attenuation of the baroreceptor reflex observed in SHR.

Mechanisms of the ventrolateral medulla on the control of sympathetic activity by central chemoreflex in rats

In the absence of inputs from carotid bodies and cardiopulmonary receptors hypercapnia markedly increases sympathetic nerve discharge (SND) to the heart and blood vessels. In the present study, splanchnic sympathetic nerve discharge (sSND) and phrenic nerve discharge (PND) were recorded in urethane anaesthetized sino‐aortic denervated and vagotomized rats. Hypercapnia (end‐expiratory CO2 from 5% to 10%) increased sSND (δ= 103 ± 7%). Bilateral injection of muscimol (GABA‐A agonist ‐ 2 mM in 30 nl) into the retrotrapezoid nucleus (RTN) eliminated PND and reduced the effect of CO2 on sSND (δ= 56 ± 8%). Injection of AP‐5 (NMDA glutamate receptor antagonist ‐ 100 mM in 50 nl) or DNQX (non NMDA glutamate receptor antagonist ‐ 100 mM in 50 nl) bilaterally into rostroventrolateral medulla (RVLM) reduced PND, but did not change the effect of CO2 on sSND. Bilateral injection of MCPG (metabotropic glutamate receptor antagonist ‐ 100 mM in 50 nL) into the RVLM reduced PND and the effect of CO2 on sSND (δ= 41 ± 7%). In conclusion, RVLM metabotropic receptors and RTN are involved in the increase in sSND produced by CO2.

Inhibition of the commissural nucleus of the solitary tract inhibits peripheral but not central chemoreflex in rats

A large variety of sensory afferents converges to the nucleus of the solitary tract (NTS) and influences the activity of ventrolateral medullary (VLM) neurons involved in cardiovascular and respiratory regulations. Here, we investigated the effects of inhibition of the commissural NTS (commNTS) on cardiovascular and respiratory responses to chemoreflex activation in conscious and anesthetized male Holtzman rats (280‐300g, n = 7/group). In conscious rats, electrolytic lesions of the commNTS eliminated the increase in mean arterial pressure (MAP) (Δ= +8±4, vs. sham: +45±18 mmHg) and reduced the increase in ventilation (404±43, vs. sham: 773±28 ml/min/kg) produced by activation of peripheral chemoreflex with KCN (40 μg/rat) intravenously (iv). However, electrolytic lesion of the commNTS did not change the increase in ventilation produced by activation of central chemoreceptors with increased levels of CO2 (7%) (1189±32, vs. sham: 1055±53 ml/min/kg). In urethane anesthetized animals, the injection of the GABA‐A agonist muscimol (2 mM ‐ 60 nL) into the commNTS eliminated the increase in MAP (Δ= +6±3, vs. saline: +23±18 mmHg), on phrenic nerve discharge (PND) and on splanchnic sympathetic nerve discharge (sSND). Muscimol into the commNTS did not change the activation of central chemoreceptors with CO2 (7%) on PND and sSND. In conclusion, the integrity of commNTS is important for the cardiorespiratory responses elicited by peripheral but not central chemoreflex activation.

The role of NMDA and non-NMDA receptors in the NTS in mediating three distinct sympathoinhibitory reflexes

Cholecystokinin (CCK) elicits a sympathetic vasomotor reflex that is implicated in gastrointestinal circulatory control. We sought to determine (1) the site in the solitary tract nucleus (NTS) responsible for mediating this reflex and (2) the possible involvement of excitatory amino acid (EAA) receptors. In addition, we sought to determine whether the NTS site responsible for mediating the baroreflex (phenylephrine, PE, 10 microg/kg i.v.) and the von Bezold-Jarisch reflex (phenylbiguanide, PBG, 10 microg/kg i.v) overlap with that involved in the CCK-induced reflex (CCK, 4 microg/kg, i.v.), and to compare the relative importance of NMDA and non-NDMA receptors in these reflexes. In separate experiments, the effects of PE, PBG, and CCK on mean arterial blood pressure, heart rate, and splanchnic sympathetic nerve discharge were tested before and after bilateral microinjection into the NTS of the gamma-aminobutyric acid(A) (GABA(A)) agonist muscimol, the EAA antagonist kynurenate, the NMDA receptor antagonist D: (-)-2-amino-5-phosphopentanoic acid (AP-5), the non-NMDA receptor antagonist 1,2,3,4-tetrahydro-6-nitro-2,3-dioxo-benzo[f]quinoxaline-7-sulfonamide (NBQX), AP-5 + NBQX, or vehicle. While all treatments (except vehicle) significantly attenuated/abolished/reversed the splanchnic sympathoinhibitory responses to PE, PBG, and CCK, the extent of blockade varied between the different treatment groups. Both NMDA and non-NMDA receptors were essential to the baroreflex and the von Bezold-Jarisch reflex, whereas the CCK reflex was more dependent on non-NMDA receptors. Muscimol, kynurenate, and AP-5 + NBQX significantly attenuated the bradycardic responses to PE and PBG (P < 0.05), whereas AP-5, NBQX, or vehicle did not. The bradycardic responses to CCK remained intact after all treatments. These results suggest that while there is overlap in the area of the NTS responsible for eliciting all three reflexes, NMDA and non-NMDA receptors are recruited differentially for the full expression of these reflexes. The CCK-induced sympathoinhibitory reflex is unique in that it relies predominantly on non-NMDA receptors in the NTS and elicits bradycardic effects that are independent of the NTS.

Biophysical and histological determinants underlying natural firing behaviors of splanchnic sympathetic preganglionic neurons in neonatal rats

Isolated thoracic spinal cords of neonatal rats spontaneously generate splanchnic sympathetic nerve discharge (SND) with a quasiperiodic rhythm ∼1-Hz. Using in vitro nerve-cord preparations that retained T6–T12 spinal segments, we investigated whether the natural firing behavior of sympathetic preganglionic neurons (SPNs) encoded the SND rhythm and what were the main biophysical and histological determinants of SPN firing. Under extracellular recording conditions, electrical stimulation of splanchnic nerves elicited antidromic responses in 212 SPNs. Among them, 92 SPNs were quiescent; 120 active SPNs had an average firing rate of 0.72±0.04 Hz, which was close to the quasiperiodic rhythm of SND. SPNs with rhythmic burst firing were rare. Probability plots of interspike intervals were constructed to extract mathematical features underlying SPN firing. Most active SPNs (88%) had a firing well described by unimodal Gaussian, suggesting a predominantly tonic pattern with normal variations. Biophysical properties of 112 SPNs were measured under whole-cell recording conditions. The charging time constant, τ, is positively correlated with the average firing rate. Histological properties were examined in 45 SPNs with intracellular diffusion of Lucifer Yellow or biocytin. SPNs with pyramidal somata and multipolar dendrites tend to be spontaneously active. In contrast, those with bipolar somata and fewer dendritic branches were quiescent in firing. These observations suggest that activity levels of SPNs are correlated with their capacity for temporal and spatial summation of synaptic inputs. How the seemingly tonic firing of individual SPNs is integrated into whole-nerve SND with quasiperiodic rhythms is discussed.

NMDA receptors in the nucleus of the solitary tract contribute to the splanchnic sympathoinhibitory effects of cholecystokinin

Cholecystokinin (CCK) elicits a sympathetic vasomotor reflex that may play an important role in regulating gastrointestinal blood flow. The aim of this study was to identify the significance of excitatory amino acid (EAA) receptors in the nucleus of the solitary tract (NTS) in this reflex. We recorded arterial blood pressure, heart rate and splanchnic sympathetic nerve discharge (SSND) in halothane anesthetised, paralysed, male Sprague-Dawley rats. In separate groups of rats, the sympathoinhibitory effects of baroreflex activation (phenylephrine, PE; 10 μg/kg i.v.), von Bezold-Jarisch reflex activation (phenylbiguanide, PBG; 10 μg/kg i.v.) and CCK (4 μg/kg, i.v.) were tested before and after bilateral microinjection into the NTS of (i) the GABAA agonist muscimol (1.75mM), (ii) the EAA receptor antagonist kynurenate (55mM) and (iii) the competitive NMDA receptor antagonist AP-5 (40mM). All treatments attenuated or completely abolished the sympathoinhibitory responses to PE, PBG and CCK. Bilateral microinjection of artificial cerebrospinal fluid into the NTS was ineffective. These results indicate (i) that the NTS neurons that mediate baroreflex- and von Bezold-Jarisch reflex-sympathoinhibition overlap with those responsible for CCK-induced sympathoinhibition and (ii) both NMDA and non-NMDA receptors in the NTS are important for all three reflexes.

The sympathoinhibitory effects of systemic cholecystokinin are dependent on neurons in the caudal ventrolateral medulla in the rat

The gastrointestinal hormone CCK inhibits a subset of presympathetic neurons in the rostroventrolateral medulla (RVLM) that may be responsible for driving the sympathetic vasomotor outflow to the gastrointestinal circulation. We tested the hypothesis that the central neurocircuitry of this novel sympathoinhibitory reflex involves a relay in the caudal ventrolateral medullary (CVLM) depressor area. Blood pressure and greater splanchnic sympathetic nerve discharge (SSND) or lumbar sympathetic nerve discharge (LSND) were monitored in anesthetised, paralyzed male Sprague-Dawley rats. The effects of phenylephrine (PE, 10 microg/kg iv; baroreflex activation), phenylbiguanide (PBG, 10 microg/kg iv; von Bezold-Jarisch reflex) and CCK (4 or 8 microg/kg iv) on SSND or LSND, were tested before and after bilateral injection of 50-100 nl of the GABAA agonist muscimol (1.75 mM; n=6, SSND; n=7, LSND) or the excitatory amino acid antagonist kynurenate (55 mM; n=7, SSND) into the CVLM. PE and PBG elicited splanchnic and lumbar sympathoinhibitory responses that were abolished by bilateral muscimol or kynurenate injection into the CVLM. Similarly, the inhibitory effect of CCK on SSND was abolished after neuronal inhibition within the CVLM. In contrast, CCK-evoked lumbar sympathoexcitation was accentuated following bilateral CVLM inhibition. In control experiments (n=7), these agents were injected outside the CVLM and had no effect on splanchnic sympathoinhibitory responses to PE, PBG, and CCK. In conclusion, neurons in the CVLM are necessary for the splanchnic but not lumbar sympathetic vasomotor reflex response to CCK. This strengthens the view that subpopulations of RVLM neurons supply sympathetic vasomotor outflow to specific vascular territories.