Systemic pro-inflammatory response facilitates the development of cerebral edema during short hypoxia.

BACKGROUND: High-altitude cerebral edema (HACE) is the severe type of acute mountain sickness (AMS) and life threatening. A subclinical inflammation has been speculated, but the exact mechanisms underlying the HACE are not fully understood. METHODS: Human volunteers ascended to high altitude (3860 m, 2 days), and rats were exposed to hypoxia in a hypobaric chamber (5000 m, 2 days). Human acute mountain sickness was evaluated by the Lake Louise Score (LLS), and plasma corticotrophin-releasing hormone (CRH) and cytokines TNF-α, IL-1ß, and IL-6 were measured in rats and humans. Subsequently, rats were pre-treated with lipopolysaccharide (LPS, intraperitoneal (ip) 4 mg/kg, 11 h) to induce inflammation prior to 1 h hypoxia (7000 m elevation). TNF-α, IL-1ß, IL-6, nitric oxide (NO), CRH, and aquaporin-4 (AQP4) and their gene expression, Evans blue, Na(+)-K(+)-ATPase activity, p65 translocation, and cell swelling were measured in brain by ELISA, Western blotting, Q-PCR, RT-PCR, immunohistochemistry, and transmission electron micrography. MAPKs, NF-κB pathway, and water permeability of primary astrocytes were demonstrated. All measurements were performed with or without LPS challenge. The release of NO, TNF-α, and IL-6 in cultured primary microglia by CRH stimulation with or without PDTC (NF-κB inhibitor) or CP154,526 (CRHR1 antagonist) were measured. RESULTS: Hypobaric hypoxia enhanced plasma TNF-α, IL-1ß, and IL-6 and CRH levels in human and rats, which positively correlated with AMS. A single LPS injection (ip, 4 mg/kg, 12 h) into rats increased TNF-α and IL-1ß levels in the serum and cortex, and AQP4 and AQP4 mRNA expression in cortex and astrocytes, and astrocyte water permeability but did not cause brain edema. However, LPS treatment 11 h prior to 1 h hypoxia (elevation, 7000 m) challenge caused cerebral edema, which was associated with activation of NF-κB and MAPKs, hypoxia-reduced Na(+)-K(+)-ATPase activity and blood-brain barrier (BBB) disruption. Both LPS and CRH stimulated TNF-α, IL-6, and NO release in cultured rat microglia via NF-κB and cAMP/PKA. CONCLUSIONS: Preexisting systemic inflammation plus a short severe hypoxia elicits cerebral edema through upregulated AQP4 and water permeability by TLR4 and CRH/CRHR1 signaling. This study revealed that both infection and hypoxia can cause inflammatory response in the brain. Systemic inflammation can facilitate onset of hypoxic cerebral edema through interaction of astrocyte and microglia by activation of TLR4 and CRH/CRHR1 signaling. Anti-inflammatory agents and CRHR1 antagonist may be useful for prevention and treatment of AMS and HACE.

Diving and exercise: the interaction of trigeminal receptors and muscle metaboreceptors on muscle sympathetic nerve activity in humans.

Swimming involves muscular activity and submersion, creating a conflict of autonomic reflexes elicited by the trigeminal receptors and skeletal muscle afferents. We sought to determine the autonomic cardiovascular responses to separate and concurrent stimulation of the trigeminal cutaneous receptors and metabolically sensitive skeletal muscle afferents (muscle metaboreflex). In eight healthy men (30 ± 2 yr) muscle sympathetic nerve activity (MSNA; microneurography), mean arterial pressure (MAP; Finometer), femoral artery blood flow (duplex Doppler ultrasonography), and femoral vascular conductance (femoral artery blood flow/MAP) were assessed during the following three experimental conditions: 1) facial cooling (trigeminal nerve stimulation), 2) postexercise ischemia (PEI; muscle metaboreflex activation) following isometric handgrip, and 3) trigeminal nerve stimulation with concurrent PEI. Trigeminal nerve stimulation produced significant increases in MSNA total activity (Δ347 ± 167%) and MAP (Δ21 ± 5%) and a reduction in femoral artery vascular conductance (Δ-17 ± 9%). PEI also evoked significant increases in MSNA total activity (Δ234 ± 83%) and MAP (Δ36 ± 4%) and a slight nonsignificant reduction in femoral artery vascular conductance (Δ-9 ± 12%). Trigeminal nerve stimulation with concurrent PEI evoked changes in MSNA total activity (Δ341 ± 96%), MAP (Δ39 ± 4%), and femoral artery vascular conductance (Δ-20 ± 9%) that were similar to those evoked by either separate trigeminal nerve stimulation or separate PEI. Thus, excitatory inputs from the trigeminal nerve and metabolically sensitive skeletal muscle afferents do not summate algebraically in eliciting a MSNA and cardiovascular response but rather exhibit synaptic occlusion, suggesting a high degree of convergent inputs on output neurons.

Switching off micturition using deep brain stimulation at midbrain sites.

Most of the time the bladder is locked in storage mode, switching to voiding only when it is judged safe and/or socially appropriate to urinate. Here we show, in humans and rodents, that deep brain stimulation in the periaqueductal gray matter can rapidly and reversibly manipulate switching within the micturition control circuitry, to defer voiding and maintain urinary continence, even when the bladder is full. Manipulation of neural continence pathways by deep brain stimulation may offer new avenues for the treatment of urinary incontinence of central origin.

Interaction between mammalian cells and Pasteurella multocida B:2. Adherence, invasion and intracellular survival.

A Pasteurella multocida B:2 strain from a case of bovine haemorrhagic septicaemia (HS) and a derivative, JRMT12, that was attenuated by a deletion in the aroA gene, were shown to adhere to, invade and survive within cultured embryonic bovine lung (EBL) cells. By comparison, bovine strains of Mannheimia haemolytica serotype A1 and P. multocida serotype A:3, although able to adhere to EBL cells, were not found intracellularly. The B:2 strains were viable intracellularly over a 7 h period, although a steady decline in viability was noted with time. Entry into the mammalian cells was inhibited by cytochalasin D, indicating that cell uptake was by an actin-dependent process. Viability assessment of EBL cells by trypan blue staining indicated that none of the bacterial strains was toxic for the EBL cells. Transmission electron microscopy (TEM) showed that, after entry into the mammalian cells, the B:2 strain resided in a vacuolar compartment. However, only a low percentage of mammalian cells appeared to contain one or more P. multocida B:2, suggesting that only certain EBL cells in the population were capable of being invaded by, or of taking up, the bacteria. TEM showed that P. multocida A:3 and M. haemolytica A:1 were found loosely adhering to the cell surface of EBL cells and were not detected intracellularly. The cell-invasive capacity of P. multocida B:2 may be a virulence property related to its ability to translocate from the respiratory tract into the blood stream.

BapC autotransporter protein is a virulence determinant of Bordetella pertussis.

A protein designated Bap-5 (GenBank accession no. AF081494) or BapC (GenBank accession no. AJ277634) has been identified as a member of the Bordetella pertussis autotransporter family and the present work suggests that this protein, like the previously characterised BrkA, is a Bvg-regulated serum resistance factor and virulence determinant. B. pertussis bapC and brkA, bapC mutants were created and, like a brkA mutant, showed greater sensitivity to killing by normal human serum than their parent strains but they were not as sensitive as a bvg mutant. Competition assays also showed an important role for BapC, like BrkA, in virulence of B. pertussis in mice after intranasal infection. Moreover, the bapC and brkA, bapC mutants, like the brkA mutant, were found to be more sensitive to the antimicrobial peptide cecropin P1 than the parent strains. In the genome sequence of B. pertussis strain Tohama, bapC is designated as a pseudogene due, in part, to a frameshift in a poly(C) tract near the 5' end of the gene which creates a truncated BapC protein. Sequence analyses of the bapC region spanning the poly(C) tract of a number of B. pertussis strains showed minor nucleotide and amino acid polymorphisms but it appeared that all had an ORF that would be able to produce BapC.

Recovery of heart rate following intense dynamic exercise.

The Olympic biathlon is a very demanding physical event that requires high oxygen delivery, good cross-country skiing skills and skilful use of a rifle. Like all high-performance endurance athletes, high cardiac vagal tone is a characteristic and extends the range over which cardiac output can increase. In the biathlete, however, the enhanced vagal control of the heart also allows a strategy for better control of stability needed for accurately firing a rifle at the end of each lap of the race. The role of endurance training, central command, reflexes from muscle, and of the carotid-cardiac baroreceptor reflex in changing vagal tone during intense exercise and recovery is discussed.

Vagus nerve stimulation inhibits the increase in Ca2+ transient and left ventricular force caused by sympathetic nerve stimulation but has no direct effects alone--epicardial Ca2+ fluorescence studies using fura-2 AM in the isolated innervated beating rabbit heart.

The effects of direct autonomic nerve stimulation on the heart may be quite different to those of perfusion with pharmacological neuromodulating agents. This study was designed to investigate the effect of autonomic nerve stimulation on intracellular calcium fluorescence using fura-2 AM in the isolated Langendorff-perfused rabbit heart preparation with intact dual autonomic innervation. The effects of autonomic nerve stimulation on cardiac force and calcium transients were more obvious during intrinsic sinus rhythm. High-frequency (15 Hz, n = 5) right vagus nerve stimulation (VS) decreased heart rate from 142.7 +/- 2.6 to 75.5 +/- 10.2 beats min(-1) and left ventricular pressure from 36.4 +/- 3.2 to 25.9 +/- 1.9 mmHg, whilst simultaneously decreasing the diastolic and systolic level of the calcium transient. Direct sympathetic nerve stimulation (7 Hz, n = 8) increased heart rate (from 144.7 +/- 10.5 to 213.2 +/- 4.9 beats min(-1)) and left ventricular pressure (from 37.5 +/- 3.6 to 43.7 +/- 2.8 mmHg), whilst simultaneously increasing the diastolic and systolic level of the calcium transient. During constant ventricular pacing, the high-frequency right vagus nerve stimulation did not have any direct effect on ventricular force or the calcium transient (n = 8), but was effective in reducing the effect of direct sympathetic nerve stimulation.

Direct evidence of nitric oxide release from neuronal nitric oxide synthase activation in the left ventricle as a result of cervical vagus nerve stimulation.

Information regarding vagal innervation in the cardiac ventricle is limited and the direct effect of vagal stimulation on ventricular myocardial function is controversial. We have recently provided indirect evidence that the anti-fibrillatory effect of vagus nerve stimulation on the ventricle is mediated by nitric oxide (NO). The aim of this study was to provide direct evidence for the release of nitric oxide in the cardiac ventricle during stimulation of the efferent parasympathetic fibres of the cervical vagus nerve. The isolated innervated rabbit heart was employed with the use of the NO fluorescent indicator 4,5-diaminofluorescein diacetate (DAF-2 DA) during stimulation of the cervical vagus nerves and acetylcholine perfusion in the absence and presence of the non-specific NO synthase inhibitor NG-nito-L-arginine (L-NNA) and the neuronal NO synthase selective inhibitor 1-(2-trifluormethylphenyl)imidazole (TRIM). Using the novel fluorescence method in the beating heart, we have shown that NO-dependent fluorescence is increased by 0.92 +/- 0.26, 1.20 +/- 0.30 and 1.91 +/- 0.27% (during low, medium and high frequency, respectively) in the ventricle in a stimulation frequency-dependent manner during vagus nerve stimulation, with comparable increases seen during separate stimulation of the left and right cervical vagus nerves. Background fluorescence is reduced during perfusion with L-NNA and the increase in fluorescence during high frequency vagal stimulation is inhibited during perfusion with both L-NNA (1.97 +/- 0.35% increase before L-NNA, 0.00 +/- 0.02% during L-NNA) and TRIM (1.78 +/- 0.18% increase before TRIM, -0.11 +/- 0.08% during TRIM). Perfusion with 0.1 microM acetylcholine increased NO fluorescence by 0.76 +/- 0.09% which was blocked by L-NNA (change of 0.00 +/- 0.03%) but not TRIM (increase of 0.82 +/- 0.21%). Activation of cardiac parasympathetic efferent nerve fibres by stimulation of the cervical vagus is associated with NO production and release in the ventricle of the rabbit, via the neuronal isoform of nitric oxide synthase.

Autonomic modulation of electrical restitution, alternans and ventricular fibrillation initiation in the isolated heart.

OBJECTIVE: Abnormal autonomic nerve activity is a strong prognostic marker for ventricular arrhythmias but the mechanisms underlying the autonomic modulation of ventricular fibrillation (VF) initiation are poorly understood. We examined the effects of direct sympathetic (SS) and vagus (VS) nerve stimulation on electrical restitution, alternans and VF threshold (VFT) in a novel isolated rabbit heart preparation with intact dual autonomic innervation. METHODS: Monophasic Action Potentials (MAPs) were recorded from a left ventricular epicardial site on innervated, isolated rabbit hearts (n=16). Standard restitution, effective refractory period (ERP), electrical alternans and VFT were measured at baseline and during SS and VS separately. RESULTS: The restitution curve was shifted downwards and made steeper with SS whilst VS caused an upward shift and a flattening of the curve. The maximum slope of restitution was increased from 1.30+/-0.10 at baseline to 1.86+/-0.17 (by 45+/-12%, P<0.01) with SS and decreased to 0.69+/-0.10 (by 51+/-6%, P<0.001) with VS. ERP was decreased from 127.3+/-2.5 ms to 111.8+/-1.8 ms with SS (by 12+/-2%, P<0.001) and increased to 144.0+/-2.2 ms with VS (by 13+/-2%, P<0.001). VFT was decreased from 4.7+/-0.6 mA to 1.9+/-0.5 mA with SS (by 64+/-5%, P<0.001) and increased to 8.7+/-1.1 mA with VS (by 89+/-14%, P<0.0005). There was a significant inverse relationship between the maximum slope of restitution and VFT (r=-0.63, P<0.0001). When compared with baseline, SS caused electrical alternans at longer pacing cycle lengths (139.0+/-8.4 vs. 123.0+/-7.8 ms, P<0.01) with greater degree of alternans (32.5+/-9.9 vs. 15.4+/-3.2%, P<0.05). It also caused a wider range of cycle lengths where alternans occurred (53.0+/-6.2 vs. 41.0+/-7.0 ms, P<0.05) whilst vagus nerve stimulation shortened this range (33.0+/-7.3 ms, P<0.001). CONCLUSIONS: Sympathetic stimulation increased maximum slope of restitution and electrical alternans but decreased ERP and VF threshold whilst vagus nerve stimulation had opposite effects. The interaction between action potential duration and beat-to-beat interval may play an important role in the autonomic modulation of VF initiation.

Central control of autonomic function.

In this review, current understanding of the control of autonomic function is outlined and its development over the last 50 years highlighted. Using the control of the cardiovascular system as the primary tool, the importance of the patterning of autonomic outflows is shown to be crucial in both homeostasis and behaviour. Technical advances have made it possible to obtain a clearer idea of how the central nervous system evolves patterns of autonomic discharge that optimise autonomic changes to support motor and behavioural responses. The specific roles of sympathetic and parasympathetic preganglionic neurones and premotor neurones are surveyed and the importance of their roles in integrating afferent inputs that result from peripheral sensory inputs and drive from multiple levels of the neuraxis is outlined. The autonomic control of the viscera, including the urinogenital organs and other organs is discussed briefly. The current ability to use animal models to monitor and modulate autonomic neural discharge and simultaneously co-relate this with end-organ activity is shown to have translational potential. There is every prospect that these studies will lead to the identification of new therapies for pathophysiological conditions.

Involvement of Corticotropin-Releasing Factor and Receptors in Immune Cells in Irritable Bowel Syndrome.

Irritable bowel syndrome (IBS) is a common functional gastrointestinal disorder defined by ROME IV criteria as pain in the lower abdominal region, which is associated with altered bowel habit or defecation. The underlying mechanism of IBS is not completely understood. IBS seems to be a product of interactions between various factors with genetics, dietary/intestinal microbiota, low-grade inflammation, and stress playing a key role in the pathogenesis of this disease. The crosstalk between the immune system and stress in IBS mechanism is increasingly recognized. Corticotropin-releasing factor (CRF), a major mediator in the stress response, is involved in altered function in GI, including inflammatory processes, colonic transit time, contractile activity, defecation pattern, pain threshold, mucosal secretory function, and barrier functions. This mini review focuses on the recently establish local GI-CRF system, its involvement in modulating the immune response in IBS, and summarizes current IBS animal models and mapping of CRF, CRFR1, and CRFR2 expression in colon tissues. CRF and receptors might be a key molecule involving the immune and movement function via brain-gut axis in IBS.

Functional selectivity of cardiac preganglionic sympathetic neurones in the rabbit heart.

BACKGROUND: Studies have shown regional and functional selectivity of cardiac postganglionic neurones indicating there might exist a similar heterogeneity in spinal segmental preganglionic neurones, which requires further investigation. METHODS: Right and left sympathetic chains were electrically stimulated from T6 to T1 in the innervated isolated rabbit heart preparation (n = 18). Sinus rate, left ventricular pressure, retrograde ventriculo-atrial conduction, monophasic action potential duration, effective refractory period, ventricular fibrillation threshold and electrical restitution were measured. RESULTS: Right sympathetic stimulation had a greater influence on heart rate (T1-T2: right; 59.9 ±â€¯6.0%, left; 41.1 ±â€¯5.6% P < 0.001) and left stimulation had greater effects on left ventricular pressure (T1-T2: right; 20.7 ±â€¯3.2%, left; 40.3 ±â€¯5.4%, P < 0.01) and ventriculo-atrial conduction (T1-T2: right; -6.8 ±â€¯1.1%, left; -15.5 ±â€¯0.2%) at all levels, with greater effects at rostral levels (T1-T3). Left sympathetic stimulation caused shorter monophasic action potentials at the base (T4-T5: right; 119.3 ±â€¯2.7 ms, left; 114.7 ±â€¯2.5 ms. P < 0.05) and apex (T4-T5: right; 118.8 ±â€¯1.2 ms, left; 114.6 ±â€¯2.6 ms. P < 0.05), greater shortening of effective refractory period (T4-T5: right; -3.6 ±â€¯1.3%, left; -7.7 ±â€¯1.8%. P < 0.05), a steeper maximum slope of restitution (T4-T5 base: right; 1.3 ±â€¯0.2, left; 1.8 ±â€¯0.2. P < 0.01. T4-T5 apex: right; 1.0 ±â€¯0.2, left; 1.6 ±â€¯0.3. P < 0.05) and a greater decrease in ventricular fibrillation threshold (T4-T5: right; -22.3 ±â€¯6.8%, left;-39.0 ±â€¯1.7%), with dominant effects at caudal levels (T4-T6). CONCLUSIONS: The preganglionic sympathetic efferent axons show functionally distinct pathways to the heart. The caudal segments (T4-T6) of the left sympathetic chain had a greater potential for arrhythmia generation and hence could pose a target for more focused clinical treatments for impairments in cardiac function.

Data highlighting the effects of spinal segmental stimulation of preganglionic sympathetic neurons on the electrophysiology of the rabbit heart.

This article presents data highlighting the functional selectivity of cardiac preganglionic sympathetic neurons in the rabbit heart. Specifically, the data draw attention to the role of each spinal segmental outflow on cardiac electrophysiology and the influence of each segment on cardiac excitability through investigating markers of arrhythmia such as electrical restitution. This data holds importance for exploring whether the preganglionic sympathetic neurons have functionally distinct pathways to the heart and whether some spinal segmental outflows have a greater potential for arrhythmia generation than others. Discussion of the data can be found in Chauhan et al. (2018) [1].

Electrophysiological effects of nicotinic and electrical stimulation of intrinsic cardiac ganglia in the absence of extrinsic autonomic nerves in the rabbit heart.

BACKGROUND: The intrinsic cardiac nervous system is a rich network of cardiac nerves that converge to form distinct ganglia and extend across the heart and is capable of influencing cardiac function. OBJECTIVE: The goals of this study were to provide a complete picture of the neurotransmitter/neuromodulator profile of the rabbit intrinsic cardiac nervous system and to determine the influence of spatially divergent ganglia on cardiac electrophysiology. METHODS: Nicotinic or electrical stimulation was applied at discrete sites of the intrinsic cardiac nerve plexus in the Langendorff-perfused rabbit heart. Functional effects on sinus rate and atrioventricular conduction were measured. Immunohistochemistry for choline acetyltransferase (ChAT), tyrosine hydroxylase, and/or neuronal nitric oxide synthase (nNOS) was performed using whole mount preparations. RESULTS: Stimulation within all ganglia produced either bradycardia, tachycardia, or a biphasic brady-tachycardia. Electrical stimulation of the right atrial and right neuronal cluster regions produced the largest chronotropic responses. Significant prolongation of atrioventricular conduction was predominant at the pulmonary vein-caudal vein region. Neurons immunoreactive (IR) only for ChAT, tyrosine hydroxylase, or nNOS were consistently located within the limits of the hilum and at the roots of the right cranial and right pulmonary veins. ChAT-IR neurons were most abundant (1946 ± 668 neurons). Neurons IR only for nNOS were distributed within ganglia. CONCLUSION: Stimulation of intrinsic ganglia, shown to be of phenotypic complexity but predominantly of cholinergic nature, indicates that clusters of neurons are capable of independent selective effects on cardiac electrophysiology, therefore providing a potential therapeutic target for the prevention and treatment of cardiac disease.

Safety and protective efficacy of intramuscular vaccination with a live aroA derivative of Pasteurella multocida B:2 against experimental hemorrhagic septicemia in calves.

Three groups of five calves, namely, V1, V2, and V3, were immunized intramuscularly at 4 and 8 weeks of age with ca. 10(9), 10(8), and 10(7) CFU, respectively, of a derivative of Pasteurella multocida B:2 wild-type strain 85020 containing a deletion in the aroA gene (strain JRMT12). The first and second vaccinations resulted in significantly (P < 0.01) higher rectal temperature responses in groups V1 and V2 than in group V3. Serum immunoglobulin M (IgM) and IgG titers did not increase in any group until after the second vaccination and were then significantly higher in groups V1 and V2 than in group V3 (P = 0.001 for both IgM and IgG). All vaccinated groups and three unvaccinated challenge control calves (group CC) were injected subcutaneously at 10 weeks of age with ca. 10(7) CFU of strain 85020. Vaccinated calves survived the challenge, but two CC animals developed clinical disease and were killed for humane reasons. After challenge, mean serum amyloid A concentrations were significantly higher (P < 0.001) in the CC group than in the vaccinated groups. Postmortem examination revealed that calves in the CC group showed the most extensive range of bacteriologically positive tissues and gross and histopathological lesions. Overall, a clear dose-dependent response was present, with those receiving a higher vaccine dose being less affected clinically, bacteriologically, and pathologically by the wild-type challenge. The V2 treatment appeared to give the best combination of high immune response, protection, and safety.

Activation of lumbosacral 5-HT2C receptors induces bursts of rhythmic activity in sympathetic nerves to the vas deferens in male rats.

1. We previously demonstrated that p-chloroamphetamine (PCA) intravenously (i.v.) evokes a specific patterned bursting response in the vas deferens nerve (VDN) of anaesthetised male rats that is associated with contraction of the vas deferens, and ejaculation and contraction of the bulbospongiosus muscles. The present study used selective 5-HT agonists to induce similar rhythmic bursting responses in the VDN in order to reveal the 5-HT receptor subtypes involved. 2. The 5-HT(2C) receptor agonist (1.0 mg kg(-1) Ro600175 i.v.) evoked the characteristic bursting pattern responses in the VDN. The 5-HT(1A) receptor agonist (1.0 mg kg(-1) 8-OH-DPAT i.v.) failed to elicit any responses. However, 8-OH-DPAT coadministered in combination with Ro600175 induced a potentiation of the responses. 3. Responses were also evoked in rats with a mid-thoracic spinalisation, with a more predictable response being observed following the combination of agonists. This suggests an action of both agonists in the lumbosacral spinal cord. 4. Responses were blocked by 0.5 mg kg(-1) SB206553 i.v. (5-HT(2B/C) receptor antagonist) or 0.5 mg kg(-1) WAY100635 i.v. (5-HT(1A) receptor antagonist), but not 0.1 or 1.0 mg kg(-1) SB269970 i.v. (5-HT(7) receptor antagonist). 5. We suggest that activation of 5-HT(2C) and 5-HT(1A) receptor subtypes synergistically elicits contraction of the vas deferens through the activation of sympathetic preganglionic neurones in the spinal cord. 6. These data support the idea of a proejaculatory action of 5-HT(2C) receptors in the lumbosacral spinal cord, suggesting a descending 5-HT excitatory pathway in addition to a 5-HT inhibitory pathway. An excitatory action of 8-OH-DPAT at lumbosacral sites is also evident.

NO-cGMP pathway at ventrolateral medullary cardiac inhibitory sites enhances the baroreceptor reflex bradycardia in the rat.

The neuronal isoform of the enzyme nitric oxide synthase (nNOS) has been identified in the caudal ventrolateral medulla of the rat close to the location of cardiac vagal motoneurones. Therefore in this study we tested identified ventral medulla cardioinhibitory sites for the involvement of nitric oxide (NO) in the baroreceptor-heart rate reflex pathway. In rats anaesthetised with a mixture of urethane (650 mg kg(-1)) and chloralose (50 mg kg(-1)) i.v., blood pressure and heart rate were monitored continuously and using stereotaxic coordinates the ventrolateral caudal brainstem within and around the nucleus ambiguus was systematically explored for sites producing a bradycardia of >50 bpm, without a change in blood pressure, using D,L homocysteic acid (DLH, 0.2 M) microinjections (50 nl) from a glass micropipette. Identified sites were marked with pontamine sky blue. Microinjection of the NO donor sodium nitroprusside (SNP, 1 mM, 50 nl) at a cardioinhibitory site also produced a significant bradycardia (68+/-14 bpm) while the NOS inhibitor N(G)-nitro-l-arginine (l-NNA) (3 mM, 50 nl) caused a small significant increase in heart rate (5+/-1 bpm). Baroreceptor reflex gain measured by the response in heart rate to a change in blood pressure induced by phenylephrine i.v. was significantly increased (610+/-171%, p<0.05) during the steady state of the response to SNP, whereas it was significantly reduced (73+/-5%, p<0.01) by l-NNA injection at a medullary cardioinhibitory site. An inhibitor of soluble guanylyl cyclase, (1)H-(1,2,4)oxadiazolo(4,3-a)quinoxalin-1-one (ODQ, 1 mM, 50 nl) also significantly reduced the baroreceptor reflex gain (63+/-8%, p<0.05). The results suggest that a NOS-cGMP signalling system in the baroreceptor reflex pathway distal to the NTS and closer to cardiac vagal motoneurones in the caudal ventral medulla contributes to enhancement of cardiac vagal tone.