Analysis of the distribution of vagal afferent projections from different peripheral organs to the nucleus of the solitary tract in rats.

Anatomical tracing studies examining the vagal system can conflate details of sensory afferent and motor efferent neurons. Here, we used a serotype of adeno-associated virus that transports retrogradely and exhibits selective tropism for vagal afferents, to map their soma location and central termination sites within the nucleus of the solitary tract (NTS). We examined the vagal sensory afferents innervating the trachea, duodenum, stomach, or heart, and in some animals, from two organs concurrently. We observed no obvious somatotopy in the somata distribution within the nodose ganglion. The central termination patterns of afferents from different organs within the NTS overlap substantially. Convergence of vagal afferent inputs from different organs onto single NTS neurons is observed. Abdominal and thoracic afferents terminate throughout the NTS, including in the rostral NTS, where the 7th cranial nerve inputs are known to synapse. To address whether the axonal labeling produced by viral transduction is so widespread because it fills axons traveling to their targets, and not just terminal fields, we labeled pre and postsynaptic elements of vagal afferents in the NTS . Vagal afferents form multiple putative synapses as they course through the NTS, with each vagal afferent neuron distributing sensory signals to multiple second-order NTS neurons. We observe little selectivity between vagal afferents from different visceral targets and NTS neurons with common neurochemical phenotypes, with afferents from different organs making close appositions with the same NTS neuron. We conclude that specific viscerosensory information is distributed widely within the NTS and that the coding of this input is probably determined by the intrinsic properties and projections of the second-order neuron.

Selective optogenetic stimulation of efferent fibers in the vagus nerve of a large mammal.

BACKGROUND: Electrical stimulation applied to individual organs, peripheral nerves, or specific brain regions has been used to treat a range of medical conditions. In cardiovascular disease, autonomic dysfunction contributes to the disease progression and electrical stimulation of the vagus nerve has been pursued as a treatment for the purpose of restoring the autonomic balance. However, this approach lacks selectivity in activating function- and organ-specific vagal fibers and, despite promising results of many preclinical studies, has so far failed to translate into a clinical treatment of cardiovascular disease. OBJECTIVE: Here we report a successful application of optogenetics for selective stimulation of vagal efferent activity in a large animal model (sheep). METHODS AND RESULTS: Twelve weeks after viral transduction of a subset of vagal motoneurons, strong axonal membrane expression of the excitatory light-sensitive ion channel ChIEF was achieved in the efferent projections innervating thoracic organs and reaching beyond the level of the diaphragm. Blue laser or LED light (>10 mW mm-2; 1 ms pulses) applied to the cervical vagus triggered precisely timed, strong bursts of efferent activity with evoked action potentials propagating at speeds of ∼6 m s-1. CONCLUSIONS: These findings demonstrate that in species with a large, multi-fascicled vagus nerve, it is possible to stimulate a specific sub-population of efferent fibers using light at a site remote from the vector delivery, marking an important step towards eventual clinical use of optogenetic technology for autonomic neuromodulation.

Sympathetic nerves control bacterial clearance.

A neural reflex mediated by the splanchnic sympathetic nerves regulates systemic inflammation in negative feedback fashion, but its consequences for host responses to live infection are unknown. To test this, conscious instrumented sheep were infected intravenously with live E. coli bacteria and followed for 48 h. A month previously, animals had undergone either bilateral splanchnic nerve section or a sham operation. As established for rodents, sheep with cut splanchnic nerves mounted a stronger systemic inflammatory response: higher blood levels of tumor necrosis factor alpha and interleukin-6 but lower levels of the anti-inflammatory cytokine interleukin-10, compared with sham-operated animals. Sequential blood cultures revealed that most sham-operated sheep maintained high circulating levels of live E. coli throughout the 48-h study period, while all sheep without splanchnic nerves rapidly cleared their bacteraemia and recovered clinically. The sympathetic inflammatory reflex evidently has a profound influence on the clearance of systemic bacterial infection.

Dexmedetomidine reduces norepinephrine requirements and preserves renal oxygenation and function in ovine septic acute kidney injury.

Norepinephrine exacerbates renal medullary hypoxia in experimental septic acute kidney injury. Here we examined whether dexmedetomidine, an α2-adrenergic agonist, can restore vasopressor responsiveness, decrease the requirement for norepinephrine and attenuate medullary hypoxia in ovine gram-negative sepsis. Sheep were instrumented with pulmonary and renal artery flow probes, and laser Doppler and oxygen-sensing probes in the renal cortex and medulla. Conscious sheep received an infusion of live Escherichia coli for 30 hours. Eight sheep in each group were randomized to receive norepinephrine, norepinephrine with dexmedetomidine, dexmedetomidine alone or saline vehicle, from 24-30 hours of sepsis. Sepsis significantly reduced the average mean arterial pressure (84 to 67 mmHg), average renal medullary perfusion (1250 to 730 perfusion units), average medullary tissue pO2 (40 to 21 mmHg) and creatinine clearance (2.50 to 0.78 mL/Kg/min). Norepinephrine restored baseline mean arterial pressure (to 83 mmHg) but worsened medullary hypoperfusion (to 330 perfusion units) and medullary hypoxia (to 9 mmHg). Dexmedetomidine (0.5 µg/kg/h) co-administration significantly reduced the norepinephrine dose (0.8 to 0.4 µg/kg/min) required to restore baseline mean arterial pressure, attenuated medullary hypoperfusion (to 606 perfusion units), decreased medullary tissue hypoxia (to 29 mmHg), and progressively increased creatinine clearance (to 1.8 mL/Kg/min). Compared with vehicle time-control, dexmedetomidine given alone significantly prevented the temporal reduction in mean arterial pressure, but had no significant effects on medullary perfusion and oxygenation or creatinine clearance. Thus, in experimental septic acute kidney injury, dexmedetomidine reduced norepinephrine requirements, attenuated its adverse effects on the renal medulla, and maintained renal function.

On the presence and functional significance of sympathetic premotor neurons with collateralized spinal axons in the rat.

KEY POINTS: Spinally-projecting neurons of the rostral ventrolateral medulla (RVLM) determine sympathetic outflow to different territories of the body. Previous studies suggest the existence of RVLM neurons with distinct functional classes, such as neurons that target sympathetic nerves bound for functionally-similar tissue types (e.g. muscle vasculature). The existence of RVLM neurons with more general actions had not been critically tested. Using viral tracing, we show that a significant minority of RVLM neurons send axon collaterals to disparate spinal segments (T2 and T10 ). Furthermore, optogenetic activation of sympathetic premotor neurons projecting to lumbar spinal segments also produced activation of sympathetic nerves from rostral spinal segments that innervate functionally diverse tissues (heart and forelimb muscle). These findings suggest the existence of individual RVLM neurons for which the axons branch to drive sympathetic preganglionic neurons of more than one functional class and may be able to produce global changes in sympathetic activity. ABSTRACT: We investigate the extent of spinal axon collateralization of rat rostral ventrolateral medulla (RVLM) sympathetic premotor neurons and its functional consequences. In anatomical tracing experiments, two recombinant herpes viral vectors with retrograde tropism and expressing different fluorophores were injected into the intermediolateral column at upper thoracic and lower thoracic levels. Histological analysis revealed that ∼21% of RVLM bulbospinal neurons were retrogradely labelled by both vectors, indicating substantial axonal collateralization to disparate spinal segments. In functional experiments, another virus with retrograde tropism, a canine adenovirus expressing Cre recombinase, was injected into the left intermediolateral horn around the thoracolumbar junction, whereas a Cre-dependent viral vector encoding Channelrhodopsin2 under LoxP control was injected into the ipsilateral RVLM. In subsequent terminal experiments, blue laser light (473 nm × 20 ms pulses at 10 mW) was used to activate RVLM neurons that had been transduced by both vectors. Stimulus-locked activation, at appropriate latencies, was recorded in the following pairs of sympathetic nerves: forelimb and hindlimb muscle sympathetic fibres, as well as cardiac and either hindlimb muscle or lumbar sympathetic nerves. The latter result demonstrates that axon collaterals of lumbar-projecting RVLM neurons project to, and excite, both functionally similar (forelimb and hindlimb muscle) and functionally dissimilar (lumbar and cardiac) preganglionic neurons. Taken together, these findings show that the axons of a significant proportion of RVLM neurons collateralise widely within the spinal cord, and that they may excite preganglionic neurons of more than one functional class.

An arterially perfused brainstem preparation of guinea pig to study central mechanisms of airway defense.

The perfused working heart brainstem preparation of rodents has become a widely used tool to study brainstem function. Here, we adapt this experimental technique for newborn guinea pigs (postnatal day 7-14) to develop a tool that enables investigation of airway defense mechanisms not observed in other rodents. The perfused guinea pig brainstem preparation generates a stable eupnea-like motor pattern recorded from the phrenic, recurrent laryngeal and intercostal nerves and basic cardio-respiratory reflexes, including the arterial chemoreceptor, the baroreceptor reflex. In addition a fictive laryngeal cough reflex can be reliably elicited after mechanical stimulation of the trachea. Single unit recordings within the ponto-medullary respiratory column show robust central respiratory neuronal activity. Additionally, as in other species ponto-medullary transection of the brainstem produces apneusis. The latter suggests that the preparation fully preserves ponto-medullary synaptic connectivity that is required for eupnea-like respiratory rhythm and pattern formation and the mediation of various cardio-respiratory reflexes. We conclude that this novel research tool provides an alternative to established rat and mouse preparations and may become a experimental tool for the investigation of central mechanisms that mediate laryngeal cough.

Anti-inflammatory reflex action of splanchnic sympathetic nerves is distributed across abdominal organs.

The splanchnic anti-inflammatory pathway has been proposed as the efferent arm of the inflammatory reflex. Although much evidence points to the spleen as the principal target organ where sympathetic nerves inhibit immune function, a systematic study to locate the target organ(s) of the splanchnic anti-inflammatory pathway has not yet been made. In anesthetized rats made endotoxemic with lipopolysaccharide (LPS, 60 µg/kg iv), plasma levels of tumor necrosis factor-α (TNF-α) were measured in animals with cut (SplancX) or sham-cut (Sham) splanchnic nerves. We confirm here that disengagement of the splanchnic anti-inflammatory pathway in SplancX rats (17.01 ± 0.95 ng/ml, mean ± SE) strongly enhances LPS-induced plasma TNF-α levels compared with Sham rats (3.76 ± 0.95 ng/ml). In paired experiments, the responses of SplancX and Sham animals were compared after the single or combined removal of organs innervated by the splanchnic nerves. Removal of target organ(s) where the splanchnic nerves inhibit systemic inflammation should abolish any difference in LPS-induced plasma TNF-α levels between Sham and SplancX rats. Any secondary effects of extirpating organs should apply to both groups. Surprisingly, removal of the spleen and/or the adrenal glands did not prevent the reflex splanchnic anti-inflammatory action nor did the following removals: spleen + adrenals + intestine; spleen + intestine + stomach and pancreas; or spleen + intestine + stomach and pancreas + liver. Only when spleen, adrenals, intestine, stomach, pancreas, and liver were all removed did the difference between SplancX and Sham animals disappear. We conclude that the reflex anti-inflammatory action of the splanchnic nerves is distributed widely across abdominal organs.

Brainstem sources of cardiac vagal tone and respiratory sinus arrhythmia.

KEY POINTS: Cardiac vagal tone is a strong predictor of health, although its central origins are unknown. Respiratory-linked fluctuations in cardiac vagal tone give rise to respiratory sinus arryhthmia (RSA), with maximum tone in the post-inspiratory phase of respiration. In the present study, we investigated whether respiratory modulation of cardiac vagal tone is intrinsically linked to post-inspiratory respiratory control using the unanaesthetized working heart-brainstem preparation of the rat. Abolition of post-inspiration, achieved by inhibition of the pontine Kolliker-Fuse nucleus, removed post-inspiratory peaks in efferent cardiac vagal activity and suppressed RSA, whereas substantial cardiac vagal tone persisted. After transection of the caudal pons, part of the remaining tone was removed by inhibition of nucleus of the solitary tract. We conclude that cardiac vagal tone depends upon at least 3 sites of the pontomedullary brainstem and that a significant proportion arises independently of RSA. ABSTRACT: Cardiac vagal tone is a strong predictor of health, although its central origins are unknown. The rat working heart-brainstem preparation shows strong cardiac vagal tone and pronounced respiratory sinus arrhythmia. In this preparation, recordings from the cut left cardiac vagal branch showed efferent activity that peaked in post-inspiration, ∼0.5 s before the cyclic minimum in heart rate (HR). We hypothesized that respiratory modulation of cardiac vagal tone and HR is intrinsically linked to the generation of post-inspiration. Neurons in the pontine Kölliker-Fuse nucleus (KF) were inhibited with bilateral microinjections of isoguvacine (50-70 nl, 10 mm) to remove the post-inspiratory phase of respiration. This also abolished the post-inspiratory peak of cardiac vagal discharge (and cyclical HR modulation), although a substantial level of activity remained. In separate preparations with intact cardiac vagal branches but sympathetically denervated by thoracic spinal pithing, cardiac chronotropic vagal tone was quantified by HR compared to its final level after systemic atropine (0.5 µm). Bilateral KF inhibition removed 88% of the cyclical fluctuation in HR but, on average, only 52% of the chronotropic vagal tone. Substantial chronotropic vagal tone also remained after transection of the brainstem through the caudal pons. Subsequent bilateral isoguvacine injections into the nucleus of the solitary tract further reduced vagal tone: remaining sources were untraced. We conclude that cardiac vagal tone depends on neurons in at least three sites of the pontomedullary brainstem, and much of it arises independently of respiratory sinus arrhythmia.

Regulation of cough by neuronal Na(+)-K(+) ATPases.

The Na(+)-K(+) ATPases play an essential role in establishing the sodium gradients in excitable cells. Multiple isoforms of the sodium pumps have been identified, with tissue and cell specific expression patterns. Because the vagal afferent nerves regulating cough must be activated at sustained high frequencies of action potential patterning to achieve cough initiation thresholds, it is a certainty that sodium pump function is essential to maintaining cough reflex sensitivities in health and in disease. The mechanisms by which Na(+)-K(+) ATPases regulate bronchopulmonary vagal afferent nerve excitability are reviewed as are potential therapeutic strategies targeting the sodium pumps in cough.

Segmental origins of cardiac sympathetic nerve activity in rats.

The segmental origins of cardiac sympathetic nerve activity (CSNA) were investigated in 8 urethane-anesthetized, artificially ventilated rats. The left upper thoracic sympathetic chain was exposed retropleurally after removing the heads of the second to fourth ribs. The preganglionic inputs to the chain from segments T1-T3 and the trunk distal to T3 were marked for later sectioning. CSNA was recorded conventionally, amplified, rectified and smoothed. Its mean level was quantified before and after each preganglionic input was cut, usually in rostro-caudal sequence. The level after all inputs were cut (i.e. noise and residual ECG pickup) was subtracted from previous measurements. The signal decrement from cutting each preganglionic input was then calculated as a percentage. CSNA in all rats depended on preganglionic drive from two or more segments, which were not always contiguous. Over the population, most preganglionic drive came from T3 and below, while the least came from T1. But there was striking inter-individual variation, such that the strongest drive to CSNA in any one rat could come from T1, T2, T3, or below T3. These findings provide new functional data on the segmental origins of CSNA in rats.

The midbrain periaqueductal grey has no role in the generation of the respiratory motor pattern, but provides command function for the modulation of respiratory activity.

It has previously been shown that stimulation of cell-columns in the periaqueductal grey (PAG) triggers site-specific cardiorespiratory effects. These are believed to facilitate changes in behaviour through coordinated changes in autonomic outflow. Here, we investigated whether PAG-evoked respiratory commands can be studied in situ using the decerebrate perfused brainstem preparation. Phrenic, vagus and abdominal iliohypogastric nerves were recorded before and after microinjection of L-glutamate (30-50 nl, 10 mM) or isoguvacine (GABA-receptor agonist, 30-50 nl, 10 mM) into the PAG. L-glutamate microinjection triggered a range of site-specific respiratory modulations (n = 17 preparations). Subsequent microinjection of isoguvacine into the same PAG sites had no effect on the baseline respiratory motor pattern or rhythm. We conclude that while the PAG has no function in respiratory pattern generation, PAG-evoked respiratory modulations can be evoked in situ in the absence of higher brain centres and while homeostatic parameters that may affect respiratory drive are held static.

Soluble receptor for advanced glycation end products (sRAGE) attenuates haemodynamic changes to chronic hypoxia in the mouse.

The calgranulin-like protein MTS1/S100A4 and the receptor for advanced glycation end-products (RAGE) have recently been implicated in mediating pulmonary arterial smooth muscle cell proliferation and vascular remodelling in experimental pulmonary arterial hypertension (PH). Here, the effects of RAGE antagonism upon 2 weeks of hypobaric hypoxia (10% O2)-induced PH in mice were assessed. Treatment with sRAGE was protective against hypobaric hypoxia-induced increases in right ventricular pressure but distal pulmonary vascular remodelling was unaffected. Intralobar pulmonary arteries from hypobaric hypoxic mice treated with sRAGE showed protection against a hypoxia-induced reduction in compliance. However, a combination of sRAGE and hypoxia also dramatically increased the force of contractions to KCl and 5-HT observed in these vessels. The acute addition of sRAGE to the organ bath produced a small, sustained contraction in intralobar pulmonary vessels and produced a synergistic enhancement of the maximal force of contraction in subsequent concentration-response curves to 5-HT. sRAGE had no effect on 5-HT-induced proliferation of Chinese hamster lung fibroblasts (CCL39), used since they have a similar pharmacological profile to mouse pulmonary fibroblasts but, surprisingly, produced a marked increase in hypoxia-induced proliferation. These data implicate RAGE as a modulator of both vasoreactivity and of proliferative processes in the response of the pulmonary circulation to chronic-hypoxia.

RAGE, vascular tone and vascular disease.

Evidence provided by both clinical and pre-clinical studies regarding a central involvement of the receptor for advanced glycation endproducts (RAGE) in vascular disease continues to mount. RAGE is upregulated as a consequence of activation of the ubiquitous pro-inflammatory transcription factor NF-kappaB which is activated in response to diverse inflammatory stimuli including hyperglycaemia, oxidised low density lipoprotein (oxLDL) and reduced shear stress. RAGE may maintain and amplify inflammatory responses in the vasculature if ligand for the receptor is present. RAGE binding by circulating advanced glycation endproducts (AGEs) or S100 protein released by activated leukocytes results in the generation of reactive oxygen species (ROS) and further activation of NF-kappaB. This leads to upregulation of adhesion molecules for circulating monocytes as well as further upregulation of RAGE itself. In addition, these ROS may scavenge and reduce bioavailability of the labile vasodilator nitric oxide (NO), reducing its anti-inflammatory effects and possibly compromising control of vascular tone directly. In addition to atherosclerosis and vascular diseases associated with diabetes, recent data from studies in transgenic mice overexpressing the RAGE ligand S100A4/MTS1 suggest a role for RAGE in the pathogenesis of pulmonary arterial hypertension (PAH). RAGE antagonism also prevents proliferation and migration of pulmonary arterial smooth muscle cells in response to 5-HT, suggesting that S100-RAGE signalling may be of key importance in pulmonary vascular homeostasis and/or disease. Further study of the role of RAGE in inflammation seems likely to yield, not only promising therapeutics but key insights into the pathophysiology of vascular disease as well.