Platinum accumulation in the brain and alteration in the central regulation of cardiovascular and respiratory functions in oxaliplatin-treated rats.

Oxaliplatin is a platinum-based alkylating chemotherapeutic agent used for cancer treatment. Neurotoxicity is one of its major adverse effects that often demands dose limitation. However, the effects of chronic oxaliplatin on the toxicity of the autonomic nervous system regulating cardiorespiratory function and adaptive reflexes are unknown. Male Sprague Dawley rats were treated with intraperitoneal oxaliplatin (3 mg kg-1 per dose) 3 times a week for 14 days. The effects of chronic oxaliplatin treatment on baseline mean arterial pressure (MAP); heart rate (HR); splanchnic sympathetic nerve activity (sSNA); phrenic nerve activity (PNA) and its amplitude (PNamp) and frequency (PNf); and sympathetic reflexes were investigated in anaesthetised, vagotomised and artificially ventilated rats. The same parameters were evaluated after acute oxaliplatin injection, and in the chronic treatment group following a single dose of oxaliplatin. The amount of platinum in the brain was determined with atomic absorption spectrophotometry. Chronic oxaliplatin treatment significantly increased MAP, sSNA and PNf and decreased HR and PNamp, while acute oxaliplatin had no effects. Platinum was accumulated in the brain after chronic oxaliplatin treatment. In the chronic oxaliplatin treatment group, further administration of a single dose of oxaliplatin increased MAP and sSNA. The baroreceptor sensitivity and somatosympathetic reflex were attenuated at rest while the sympathoexcitatory response to hypercapnia was increased in the chronic treatment group. This is the first study to reveal oxaliplatin-induced alterations in the central regulation of cardiovascular and respiratory functions as well as reflexes that may lead to hypertension and breathing disorders which may be mediated via accumulated platinum in the brain.

Local ionotropic glutamate receptors are required to trigger and sustain ramping of sympathetic nerve activity by hypothalamic paraventricular nucleus TNFα.

Tumor necrosis factor-α (TNFα) in the hypothalamic paraventricular nucleus (PVN) contributes to increased sympathetic nerve activity (SNA) in cardiovascular disease models, but mechanisms are incompletely understood. As previously reported, bilateral PVN TNFα (0.6 pmol, 50 nL) induced acute ramping of splanchnic SNA (SSNA) that averaged +64 ± 7% after 60 min and +109 ± 17% after 120 min (P < 0.0001, n = 10). Given that TNFα can rapidly strengthen glutamatergic transmission, we hypothesized that progressive activation of ionotropic glutamate receptors is critically involved. When compared with that of vehicle (n = 5), prior blockade of PVN AMPA or NMDA receptors in anesthetized (urethane/α-chloralose) adult male Sprague-Dawley rats dose-dependently (ED50: 2,3-dioxo-6-nitro-1,2,3,4-tetrahydrobenzo[f]quinoxaline-7-sulfonamide (NBQX), 2.48 nmol; D-(-)-2-amino-5-phosphonopentanoic acid (APV), 12.33 nmol), but incompletely (Emax: NBQX, 64%; APV, 41%), attenuated TNFα-induced SSNA ramping (n = 5/dose). By contrast, combined receptor blockade prevented ramping (1.3 ± 2.1%, P < 0.0001, n = 5). Whereas separate blockade of PVN AMPA or NMDA receptors (n = 5/group) had little effect on continued SSNA ramping when performed 60 min after TNFα injection, combined blockade (n = 5) or PVN inhibition with the GABA-A receptor agonist muscimol (n = 5) effectively stalled, without reversing, the SSNA ramp. Notably, PVN TNFα increased local TNFα immunofluorescence after 120, but not 60 min. Findings indicate that AMPA and NMDA receptors each contribute to SSNA ramping to PVN TNFα, and that their collective availability and ongoing activity are required to initiate and sustain the ramping response. We conclude that acute sympathetic activation by PVN TNFα involves progressive local glutamatergic excitation that recruits downstream neurons capable of maintaining heightened SSNA, but incapable of sustaining SSNA ramping.NEW & NOTEWORTHY The proinflammatory cytokine TNFα contributes to heightened SNA in cardiovascular disease models, but mechanisms remain obscure. Here, we demonstrate that TNFα injection into the hypothalamic PVN triggers SNA ramping by mechanisms dependent on local ionotropic glutamate receptor availability, but largely independent of TNFα autoinduction. Continued SNA ramping depends on ionotropic glutamate receptor and neuronal activity in PVN, indicating that strengthening and/or increased efficacy of glutamatergic transmission is necessary for acute sympathoexcitation by PVN TNFα.

Neurolytic Splanchnic Nerve Block and Pain Relief, Survival, and Quality of Life in Unresectable Pancreatic Cancer: A Randomized Controlled Trial.

BACKGROUND: Neurolytic splanchnic nerve block is used to manage pancreatic cancer pain. However, its impact on survival and quality of life remains controversial. The authors' primary hypothesis was that pain relief would be better with a nerve block. Secondarily, they hypothesized that analgesic use, survival, and quality of life might be affected. METHODS: This randomized, double-blind, parallel-armed trial was conducted in five Chinese centers. Eligible patients suffering from moderate to severe pain conditions were randomly assigned to receive splanchnic nerve block with either absolute alcohol (neurolysis) or normal saline (control). The primary outcome was pain relief measured on a visual analogue scale. Opioid consumption, survival, quality of life, and adverse effects were also documented. Analgesics were managed using a protocol common to all centers. Patients were followed up for 8 months or until death. RESULTS: Ninety-six patients (48 for each group) were included in the analysis. Pain relief with neurolysis was greater for the first 3 months (largest at the first month; mean difference, 0.7 [95% CI, 0.3 to 1.0]; adjusted P < 0.001) compared with placebo injection. Opioid consumption with neurolysis was lower for the first 5 months (largest at the first month; mean difference, 95.8 [95% CI, 67.4 to 124.1]; adjusted P < 0.001) compared with placebo injection. There was a significant difference in survival (hazard ratio, 1.56 [95% CI, 1.03 to 2.35]; P = 0.036) between groups. A significant reduction in survival in neurolysis was found for stage IV patients (hazard ratio, 1.94 [95% CI, 1.29 to 2.93]; P = 0.001), but not for stage III patients (hazard ratio, 1.08 [95% CI, 0.59 to 1.97]; P = 0.809). No differences in quality of life were observed. CONCLUSIONS: Neurolytic splanchnic nerve block appears to be an effective option for controlling pain and reducing opioid requirements in patients with unresectable pancreatic cancer.

Seizure-Induced Sympathoexcitation Is Caused by Activation of Glutamatergic Receptors in RVLM That Also Causes Proarrhythmogenic Changes Mediated by PACAP and Microglia in Rats.

Cardiovascular autonomic dysfunction in seizure is a major cause of sudden unexpected death in epilepsy. The catecholaminergic neurons in the rostral ventrolateral medulla (RVLM) maintain sympathetic vasomotor tone and blood pressure through their direct excitatory projections to the intermediolateral (IML) cell column. Glutamate, the principal excitatory neurotransmitter in brain, is increased in seizures. Pituitary adenylate cyclase activating polypeptide (PACAP) is an excitatory neuropeptide with neuroprotective properties, whereas microglia are key players in inflammatory responses in CNS. We investigated the roles of glutamate, PACAP, and microglia on RVLM catecholaminergic neurons during the cardiovascular responses to 2 mg/kg kainic acid (KA)-induced seizures in urethane anesthetized, male Sprague Dawley rats. Microinjection of the glutamate antagonist, kynurenic acid (50 nl; 100 mM) into RVLM, blocked the seizure-induced 43.2 ± 12.6% sympathoexcitation (p ≤ 0.05), and abolished the pressor responses, tachycardia, and QT interval prolongation. PACAP or microglia antagonists (50 nl) (PACAP(6-38), 15 pmol; minocycline 10 mg/ml) microinjected bilaterally into RVLM had no effect on seizure-induced sympathoexcitation, pressor responses, or tachycardia but abolished the prolongation of QT interval. The actions of PACAP or microglia on RVLM neurons do not cause sympathoexcitation, but they do elicit proarrhythmogenic changes. An immunohistochemical analysis in 2 and 10 mg/kg KA-induced seizure rats revealed that microglia surrounding catecholaminergic neurons are in a "surveillance" state with no change in the number of M2 microglia (anti-inflammatory). In conclusion, seizure-induced sympathoexcitation is caused by activation of glutamatergic receptors in RVLM that also cause proarrhythmogenic changes mediated by PACAP and microglia. SIGNIFICANCE STATEMENT: Sudden unexpected death in epilepsy is a major cause of death in epilepsy. Generally, seizures are accompanied by changes in brain function leading to uncontrolled nerve activity causing high blood pressure, rapid heart rate, and abnormal heart rhythm. Nevertheless, the brain chemicals causing these cardiovascular changes are unknown. Chemicals, such as glutamate and pituitary adenylate cyclase activating polypeptide, whose expression is increased after seizures, act on specific cardiovascular nuclei in the brain and influence the activity of the heart, and blood vessels. Microglia, which manage excitation in the brain, are commonly activated after seizure and produce pro- and/or anti-inflammatory factors. Hence, we aimed to determine the effects of blocking glutamate, pituitary adenylate cyclase activating polypeptide, and microglia in the RVLM and their contribution to cardiovascular autonomic dysfunction in seizure.

Impact of Chromogranin A deficiency on catecholamine storage, catecholamine granule morphology and chromaffin cell energy metabolism in vivo.

Chromogranin A (CgA) is a prohormone and granulogenic factor in neuroendocrine tissues with a regulated secretory pathway. The impact of CgA depletion on secretory granule formation has been previously demonstrated in cell culture. However, studies linking the structural effects of CgA deficiency with secretory performance and cell metabolism in the adrenomedullary chromaffin cells in vivo have not previously been reported. Adrenomedullary content of the secreted adrenal catecholamines norepinephrine (NE) and epinephrine (EPI) was decreased 30-40 % in Chga-KO mice. Quantification of NE and EPI-storing dense core (DC) vesicles (DCV) revealed decreased DCV numbers in chromaffin cells in Chga-KO mice. For both cell types, the DCV diameter in Chga-KO mice was less (100-200 nm) than in WT mice (200-350 nm). The volume density of the vesicle and vesicle number was also lower in Chga-KO mice. Chga-KO mice showed an ~47 % increase in DCV/DC ratio, implying vesicle swelling due to increased osmotically active free catecholamines. Upon challenge with 2 U/kg insulin, there was a diminution in adrenomedullary EPI, no change in NE and a very large increase in the EPI and NE precursor dopamine (DA), consistent with increased catecholamine biosynthesis during prolonged secretion. We found dilated mitochondrial cristae, endoplasmic reticulum and Golgi complex, as well as increased synaptic mitochondria, synaptic vesicles and glycogen granules in Chga-KO mice compared to WT mice, suggesting that decreased granulogenesis and catecholamine storage in CgA-deficient mouse adrenal medulla is compensated by increased VMAT-dependent catecholamine update into storage vesicles, at the expense of enhanced energy expenditure by the chromaffin cell.

Attenuation of angiotensin type 2 receptor function in the rostral ventrolateral medullary pressor area of the spontaneously hypertensive rat.

We hypothesized that blockade of angiotensin II type 2 receptors (AT2Rs) in the rostral ventrolateral medullary pressor area (RVLM) may elicit sympathoexcitatory responses which are smaller in hypertensive rats compared to normotensive rats. This hypothesis was tested in urethane-anesthetized, artificially ventilated male 14-week-old spontaneously hypertensive rats (SHR). Age-matched male Wistar-Kyoto rats (WKY) and Wistar rats were used as controls. PD123319 (AT2R antagonist) was microinjected into the RVLM and mean arterial pressure (MAP), heart rate (HR) and greater splanchnic nerve activity (GSNA) were recorded. Increases in MAP, HR and GSNA elicited by unilateral microinjections of PD123319 into the RVLM were significantly smaller in SHR when compared with those in WKY and Wistar rats. Unilateral microinjections of l-glutamate (l-Glu) into the RVLM elicited greater increases in MAP and GSNA in SHR compared to those in WKY. AT2R immunoreactivity was demonstrated in the RVLM neurons which were retrogradely labeled from the intermediolateral cell column (IML) of the spinal cord. These results indicate that AT2Rs are present on the RVLM neurons projecting to the IML and their blockade results in sympathoexcitatory responses. Activation of AT2Rs has an inhibitory influence in the RVLM and these receptors are tonically active. Attenuation of the function of AT2Rs in the RVLM may play a role in genesis and/or maintenance of hypertension in SHR.

Leptin differentially increases sympathetic nerve activity and its baroreflex regulation in female rats: role of oestrogen.

Obesity and hypertension are commonly associated, and activation of the sympathetic nervous system is considered to be a major contributor, at least in part due to the central actions of leptin. However, while leptin increases sympathetic nerve activity (SNA) in males, whether leptin is equally effective in females is unknown. Here, we show that intracerebroventricular (i.c.v.) leptin increases lumbar (LSNA) and renal (RSNA) SNA and baroreflex control of LSNA and RSNA in α-chloralose anaesthetized female rats, but only during pro-oestrus. In contrast, i.c.v. leptin increased basal and baroreflex control of splanchnic SNA (SSNA) and heart rate (HR) in rats in both the pro-oestrus and dioestrus states. The effects of leptin on basal LSNA, RSNA, SSNA and HR were similar in males and pro-oestrus females; however, i.c.v. leptin increased mean arterial pressure (MAP) only in males. Leptin did not alter LSNA or HR in ovariectomized rats, but its effects were normalized with 4 days of oestrogen treatment. Bilateral nanoinjection of SHU9119 into the paraventricular nucleus of the hypothalamus (PVN), to block α-melanocyte-stimulating hormone (α-MSH) type 3 and 4 receptors, decreased LSNA in leptin-treated pro-oestrus but not dioestrus rats. Unlike leptin, i.c.v. insulin infusion increased basal and baroreflex control of LSNA and HR similarly in pro-oestrus and dioestrus rats; these responses did not differ from those in male rats. We conclude that, in female rats, leptin's stimulatory effects on SNA are differentially enhanced by oestrogen, at least in part via an increase in α-MSH activity in the PVN. These data further suggest that the actions of leptin and insulin to increase the activity of various sympathetic nerves occur via different neuronal pathways or cellular mechanisms. These results may explain the poor correlation in females of SNA with adiposity, or of MAP with leptin.

Sympathoexcitation and pressor responses induced by ethanol in the central nucleus of amygdala involves activation of NMDA receptors in rats.

The central nervous system plays an important role in regulating sympathetic outflow and arterial pressure in response to ethanol exposure. However, the underlying neural mechanisms have not been fully understood. In the present study, we tested the hypothesis that injection of ethanol in the central nucleus of the amygdala (CeA) increases sympathetic outflow, which may require the activation of local ionotropic excitatory amino acid receptors. In anesthetized rats, CeA injection of ethanol (0, 0.17, and 1.7 µmol) increased splanchnic sympathetic nerve activity (SSNA), lumbar sympathetic nerve activity (LSNA), and mean arterial pressure (MAP) in a dose-dependent manner. A cocktail containing ethanol (1.7 µmol) and kynurenate (KYN), an ionotropic excitatory amino acid receptor blocker, showed significantly blunted sympathoexcitatory and pressor responses compared with those elicited by CeA-injected ethanol alone (P < 0.01). A cocktail containing ethanol and d-2-amino-5-phosphonovalerate, an N-methyl-d-aspartate (NMDA) receptor antagonist, elicited attenuated sympathoexcitatory and pressor responses that were significantly less than ethanol alone (P < 0.01). In addition, CeA injection of acetate (0.20 µmol, n = 7), an ethanol metabolite, consistently elicited sympathoexcitatory and pressor responses, which were effectively blocked by d-2-amino-5-phosphonovalerate (n = 9, P < 0.05). Inhibition of neuronal activity of the rostral ventrolateral medulla (RVLM) with KYN significantly (P < 0.01) attenuated sympathoexcitatory responses elicited by CeA-injected ethanol. Double labeling of immune fluorescence showed NMDA NR1 receptor expression in CeA neurons projecting to the RVLM. We conclude that ethanol and acetate increase sympathetic outflow and arterial pressure, which may involve the activation of NMDA receptors in CeA neurons projecting to the RVLM.

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.

Ageing alters perivascular nerve function of mouse mesenteric arteries in vivo.

Abstract Mesenteric arteries (MAs) are studied widely in vitro but little is known of their reactivity in vivo. Transgenic animals have enabled Ca(2+) signalling to be studied in isolated MAs but the reactivity of these vessels in vivo is undefined. We tested the hypothesis that ageing alters MA reactivity to perivascular nerve stimulation (PNS) and adrenoreceptor (AR) activation during blood flow control. First- (1A), second- (2A) and third-order (3A) MAs of pentobarbital-anaesthetized Young (3-6 months) and Old (24-26 months) male and female Cx40(BAC)-GCaMP2 transgenic mice (C57BL/6 background; positive or negative for the GCaMP2 transgene) were studied with intravital microscopy. A segment of jejunum was exteriorized and an MA network was superfused with physiological salt solution (pH 7.4, 37°C). Resting tone was 10% in MAs of Young and Old mice; diameters were ∼5% (1A), 20% (2A) and 40% (3A) smaller (P 0.05) in Old mice. Throughout MA networks, vasoconstriction increased with PNS frequency (1-16 Hz) but was ∼20% less in Young vs. Old mice (P 0.05) and was inhibited by tetrodotoxin (1 µm). Capsaicin (10 µm; to inhibit sensory nerves) enhanced MA constriction to PNS (P 0.05) by ∼20% in Young but not Old mice. Phenylephrine (an α1AR agonist) potency was greater in Young mice (P 0.05) with similar efficacy (∼60% constriction) across ages and MA branches. Constrictions to UK14304 (an α2AR agonist) were less (∼20%; P 0.05) and were unaffected by ageing. Irrespective of sex or transgene expression, ageing consistently reduced the sensitivity of MAs to α1AR vasoconstriction while blunting the attenuation of sympathetic vasoconstriction by sensory nerves. These findings imply substantive alterations in splanchnic blood flow control with ageing.

[Postoperative pancreonecrosis prophylaxis in a gastric cancer surgery].

Efficacy of a new method of intraoperative regional sympathetic blockade of splanchnic nerves was estimated, basing on the results of surgical treatment of 3134 patients, suffering gastric cancer. While comparing the immediate results of surgical interventions there was established, that the risk of postoperative pancreatitis occurrence (lethality) is dependent on the kind of operation performed and its traumaticity. The risk of postoperative pancreonecrosis occurrence (lethality) lowering after standard surgical and combined intervention, after gastric surgery with pancreatic resection was promoted by application of a spiritus-novocaine blockade with the objective to suppress sympathetic reflexes as an element of anesthesia intraoperatively and in early postoperative period.

Cardiovascular responses elicited by a new endogenous angiotensin in the nucleus tractus solitarius of the rat.

Cardiovascular effects of angiotensin-(1-12) [ANG-(1-12)] were studied in the medial nucleus of the tractus solitarius (mNTS) in anesthetized, artificially ventilated, adult male Wistar rats. Microinjections (100 nl) of ANG-(1-12) (0.06 mM) into the mNTS elicited maximum decreases in mean arterial pressure (MAP; 34 ± 5.8 mmHg) and heart rate (HR; 39 ± 3.7 beats/min). Bilateral vagotomy abolished ANG-(1-12)-induced bradycardia. Efferent greater splanchnic nerve activity was decreased by microinjections of ANG-(1-12) into the mNTS. Blockade of ANG type 1 receptors (AT(1)Rs; using ZD-7155 or L-158,809), but not ANG type 2 receptors (AT(2)Rs; using PD-123319), significantly attenuated ANG-(1-12)-induced cardiovascular responses. Simultaneous inhibition of both angiotensin-converting enzyme (ACE; using captopril) and chymase (using chymostatin) completely blocked the effects of ANG-(1-12). Microinjections of A-779 [ANG-(1-7) antagonist] did not attenuate ANG-(1-12)-induced responses. Pressure ejection of ANG-(1-12) (0.06 mM, 2 nl) caused excitation of barosensitive mNTS neurons, which was blocked by prior application of the AT(1)R antagonist. ANG-(1-12)-induced excitation of mNTS neurons was also blocked by prior sequential applications of captopril and chymostatin. These results indicate that 1) microinjections of ANG-(1-12) into the mNTS elicited depressor and bradycardic responses by exciting barosensitive mNTS neurons; 2) the decreases in MAP and HR were mediated via sympathetic and vagus nerves, respectively; 3) AT(1)Rs, but not AT(2)Rs, mediated these actions of ANG-(1-12); 4) the responses were mediated via the conversion of ANG-(1-12) to ANG II and both ACE and chymase were involved in this conversion; and 5) ANG-(1-7) was not one of the metabolites of ANG-(1-12) in the mNTS.

The hypothalamic arcuate nucleus: a new site of cardiovascular action of angiotensin-(1-12) and angiotensin II.

The hypothalamic arcuate nucleus (ARCN) has been reported to play a significant role in cardiovascular regulation. It has been hypothesized that the ARCN may be one of the sites of cardiovascular actions of angiotensins (ANGs). Experiments were carried out in urethane-anesthetized, artificially ventilated, adult male Wistar rats. The ARCN was identified by microinjections of N-methyl-d-aspartic acid (NMDA; 10 mM). Microinjections (50 nl) of ANG-(1-12) (1 mM) into the ARCN elicited increases in mean arterial pressure (MAP), heart rate (HR), and greater splanchnic nerve activity (GSNA). The tachycardic responses to ANG-(1-12) were attenuated by bilateral vagotomy. The cardiovascular responses elicited by ANG-(1-12) were attenuated by microinjections of ANG II type 1 receptor (AT(1)R) antagonists but not ANG type 2 receptor (AT(2)R) antagonist. Combined inhibition of ANG-converting enzyme (ACE) and chymase in the ARCN abolished ANG-(1-12)-induced responses. Microinjections of ANG II (1 mM) into the ARCN also increased MAP and HR. Inhibition of ARCN by microinjections of muscimol (1 mM) attenuated the pressor and tachycardic responses to intravenously administered ANG-(1-12) and ANG II (300 pmol/kg each). These results indicated that 1) microinjections of ANG-(1-12) into the ARCN elicited increases in MAP, HR, and GSNA; 2) HR responses were mediated via both sympathetic and vagus nerves; 3) AT(1)Rs, but not AT(2)Rs, in the ARCN mediated ANG-(1-12)-induced responses; 4) both ACE and chymase were needed to convert ANG-(1-12) to ANG II in the ARCN; and 5) ARCN plays a role in mediating the cardiovascular responses to circulating ANGs.

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.

Intrathecal bombesin is sympathoexcitatory and pressor in rat.

Bombesin, a 14 amino-acid peptide, is pressor when administered intravenously in rat and pressor and sympathoexcitatory when applied intracerebroventricularly. To determine the spinal effects of bombesin, the peptide was administered acutely in the intrathecal space at around thoracic spinal cord level six of urethane-anesthetized, paralyzed, and bilaterally vagotomized rats. Blood pressure, heart rate, splanchnic sympathetic nerve activity (sSNA), phrenic nerve activity, and end-tidal CO(2) were monitored to evaluate changes in the cardiorespiratory systems. Bombesin elicited a long-lasting excitation of sSNA associated with an increase in blood pressure and tachycardia. There was a mean increase in arterial blood pressure of 52 ± 5 mmHg (300 µM; P < 0.01). Heart rate and sSNA also increased by 40 ± 4 beats/min (P < 0.01) and 162 ± 33% (P < 0.01), respectively. Phrenic nerve amplitude (PNamp, 73 ± 8%, P < 0.01) and phrenic expiratory period (+0.16 ± 0.02 s, P < 0.05) increased following 300 µM bombesin. The gain of the sympathetic baroreflex increased from -2.8 ± 0.7 to -5.4 ± 0.9% (P < 0.01), whereas the sSNA range was increased by 99 ± 26% (P < 0.01). During hyperoxic hypercapnia (10% CO(2) in O(2), 90 s), bombesin potentiated the responses in heart rate (-25 ± 5 beats/min, P < 0.01) and sSNA (+136 ± 29%, P < 0.001) but reduced PNamp (from 58 ± 6 to 39 ± 7%, P < 0.05). Finally, ICI-216,140 (1 mM), an in vivo antagonist for the bombesin receptor 2, attenuated the effects of 300 µM bombesin on blood pressure (21 ± 7 mmHg, P < 0.01). We conclude that bombesin is sympathoexcitatory at thoracic spinal segments. The effect on phrenic nerve activity may the result of spinobulbar pathways and activation of local motoneuronal pools.

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.

Basal sympathetic activity generated in neonatal mouse brainstem-spinal cord preparation requires T-type calcium channel subunit 1H.

The T-type calcium channel (T-channel) is a low-voltage-activated channel. Whether T-channels are involved in sympathetic nerve discharge (SND), with subunits α1G and α1H differentially regulating SND genesis, was explored using in vitro brainstem-spinal cord-splanchnic sympathetic nerve preparations of wild-type and genetically modified B6 mice. Applications of 10-80 µm NNC 55-0396 to block T-channels in wild-type mice reduced SND in a concentration-dependent manner. Amounts of SND were measured in units of signal-to-noise ratio for objective comparisons between mouse groups. Comparable amounts of SND were observed in wild-type and α1G(-/-) mice. However, only ∼40% of the amount of SND of that in wild-type or α1G(-/-) mice was observed in α1H(-/-) mice. Whether a diminished excitatory drive originating in the brainstem could explain a low SND in α1H(-/-) mice was evaluated by cervical cord transections. Isolated spinal cord preparations of mice with different genetic backgrounds produced comparable amounts of SND. Excitability of the spinal circuitry was further explored by bath applications of 5 mm glutamate. Glutamate applications produced a prominent SND rise in all mouse groups. The ratios of glutamate-induced SND rise were similar between wild-type and α1H(-/-) mice, but significantly higher in α1G(-/-) mice. Taken together, these results suggest that α1H in mouse brainstem is essential for the genesis of presympathetic drive, whereas α1G in mouse spinal cord is functionally inhibitory for SND genesis. We conclude that α1H and α1G T-channel subunits may differentially regulate mouse SND genesis at different levels of the neuraxis.

Galanin suppresses visceral afferent responses to noxious mechanical and inflammatory stimuli.

Galanin is a neuropeptide expressed by sensory neurones innervating the gastrointestinal (GI) tract. Galanin displays inhibitory effects on vagal afferent signaling within the upper GI tract, and the goal of this study was to determine the actions of galanin on colonic spinal afferent function. Specifically, we sought to evaluate the effect of galanin on lumbar splanchnic nerve (LSN) mechanosensitivity to noxious distending pressures and the development of hypersensitivity in the presence of inflammatory stimuli and colitis. Using ex vivo electrophysiological recordings we show that galanin produces a dose-dependent suppression of colonic LSN responses to mechanical stimuli and prevents the development of hypersensitivity to acutely administered inflammatory mediators. Using galanin receptor (GalR) agonists, we show that GalR1 activation, but not GalR2/3 activation, suppresses mechanosensitivity. The effect of galanin on colonic afferent activity was not observed in tissue from mice with dextran sodium sulfate-induced colitis. We conclude that galanin has a marked suppressive effect on colonic mechanosensitivity at noxious distending pressures and prevents the acute development of mechanical hypersensitivity to inflammatory mediators, an effect not seen in the inflamed colon. These actions highlight a potential role for galanin in the regulation of mechanical nociception in the bowel and the therapeutic potential of targeting galaninergic signaling to treat visceral hypersensitivity.