AbstractIntroductionVagus nerve stimulation (VNS) is an effective treatment for people with drug‐resistant epilepsy. However, its mechanisms of action are poorly understood, including which nerve fibers are activated in humans during VNS in typical clinical settings and which are required for clinical efficacy. In particular, there have been no intraneural recordings of vagus nerve fiber activation in awake humans undergoing chronic VNS. In this study, for the first time, we report recordings from the vagus nerve in this setting.MethodsThe recordings were performed using a sterile tungsten microelectrode inserted percutaneously into the cervical vagus nerve under ultrasound guidance. The clinical VNS systems were used to deliver stimulation while activity in the vagus nerve was recorded.ResultsIn addition to activating myelinated axons at low currents, we provide evidence that VNS can also activate unmyelinated C fibers in the vagus nerve at currents <1 mA.ConclusionsThese results add to our understanding of how VNS exerts its beneficial effects in drug‐resistant epilepsy.Plain Language StatementHere we describe for the first time, electrical recordings from the vagus nerve in awake drug‐resistant epilepsy patients with an implanted vagus nerve stimulation (VNS) device. We found that the VNS device was able to activate both myelinated and unmyelinated fibers within the vagus nerve, which contributes to our understanding of how VNS works in the context of drug‐resistant epilepsy.

Microneurographic recordings of the human cervical vagus nerve have revealed the presence of multi-unit neural activity with measurable cardiac rhythmicity. This suggests that the physiology of vagal neurones with cardiovascular regulatory function can be studied using this method. Here, the activity of cardiac rhythmic single units was discriminated from human cervical vagus nerve recordings using template-based waveform matching. The activity of 44 cardiac rhythmic neurones (22 with myelinated axons and 22 with unmyelinated axons) was isolated. By consideration of each unit's firing pattern with respect to the cardiac and respiratory cycles, the functional identification of each unit was attempted. Of note is the observation of seven cardiac rhythmic neurones with myelinated axons whose activity was recruited or enhanced by slow, deep breathing, was maximal during the nadir of respiratory sinus arrhythmia, and showed an expiratory peak. This is characteristic of cardioinhibitory efferent neurones, which are responsible for respiratory sinus arrhythmia. The remaining 15 cardiac rhythmic neurones with myelinated axons were categorised as cardiopulmonary receptors or arterial baroreceptors based on the position of their peak in firing with respect to the R-wave of the cardiac cycle. This latter method is not viable for neurones with unmyelinated axons due to their slow and unknown conduction velocities. With the exception of three neurones whose expiratory modulation implicates them as cardiac-projecting efferent neurones, this population is likely dominated by arterial baroreceptors. In conclusion, the activity of single units with cardiovascular function has been discriminated within the human cervical vagus, enabling their systematic study. KEY POINTS: Recordings of the electrical activity of the vagus nerve have recently been made at the level of the neck in humans. Examination of the gross activity of this nerve reveals subpopulations of neurones whose activity fluctuates in time with the heart's beat, suggesting that the neurones that monitor or modify cardiac function can be studied using this method. Here, the activity of individual cardiac rhythmic neurones was isolated from human vagus nerve recordings using template-based spike sorting. The relationship between this activity and the cardiac and respiratory cycles was used as a means of classifying each neurone. Neuronal firing patterns that are consistent with that of neurones that modify cardiac function, including heart-slowing 'cardioinhibitory' neurones, as well as neurones that inform the brain of cardiovascular status were observed. This approach enables, for the first time, the systematic study of the function of these neurones in humans in both health and disease.

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.

The cover image is based on the Research Article Analysis of the distribution of vagal afferent projections from different peripheral organs to the nucleus of the solitary tract in rats by Jaspreet K. Bassi et al., https://doi.org/10.1002/cne.25398.

The pre-Bötzinger Complex (preBötC) is a major generator of respiratory rhythm and is crucial for respiratory modulation of orofacial behaviours. The nasolabialis profundus (NLP) is a facial muscle located in the rostral part of the rat snout that causes vibrissae protraction. NLP motoneurons receive direct input from the preBötC, but the identity of the responsible preBötC neurons is not fully elucidated. The aim of this study was to identify this preBötC neurons sub-group based on their axonal projections and physiological function. To achieve this, we developed a cre-recombinase (Cre) dependent adeno-associated virus (AAV) that expressed the light-activated chloride channel (GtACR2) predominantly in the soma (AAV-DIO-GtACR2-muGFP-Kv2.1). To validate this new AAV, it was injected with AAV-Cre-TdTomato into the preBötC of Sprague-Dawley rats (60-80 g, n=2). Three weeks later, rats were anesthetised with intravenous urethane, and blood pressure (BP), heart rate (HR), diaphragm (dEMG) and NLP electromyography (EMG) were recorded. Optical fibers were bilaterally implanted into the preBötC, and 470 nm light (50 Hz, 5 ms pulse, 20 mW) delivered for 30 s to photoinhibit neurons. The results are expressed as mean ± SD. We observed effective Cre recombination, resulting in GtACR2-muGFP expression in the preBötC. During photoinhibition of preBötC, dEMG initially ceased, but this did not last for the entire photoinhibition period (8 ± 2 s); HR decreased (ΔHR: -10 ± 1.2% from baseline); and BP decreased during apnea (ΔMAP: -10 ± 10 % from baseline), but returned toward baseline levels as breathing resumed. Under baseline conditions, activity of the NLP occurred almost exclusively during inspiration, with a limited pre-I component activity. All NLP EMG activity ceased during initial photoinhibition and then returned with activity in the dEMG. During the photoinhibition, when breathing had resumed, we observed an enlargement of the duration of the pre-I component of NLP (0.27 ± 0.02 vs. baseline: 0.06 ± 0.0004 s). To selectively inhibit preBötC neurons that project to the NLP motoneurons, we injected a retrogradely transporting AAV expressing Cre (AAVrg-mCherry-Cre) into the lateral edge of the 7n, and AAV-DIO-GtACR2-muGFP-Kv2.1 into the preBötC (n=2). Three weeks later, we performed the same experimental protocol as described above. Postmortem histology showed strong muGFP expression in a sub-group of preBötC neurons. Selective photoinhibition of these preBötC neurons that project to the 7n neurons reduced NLP activity (Δ amplitude: -42 ± 6% from baseline) and the pre-I component (0.03 ± 0.02 vs. baseline: 0.08 ± 0.001 s s), without affecting breathing, BP or HR. In conclusion, beyond validating a new optogenetic tool, our results suggest that separate populations of preBötC neurons, with coordinated respiratory rhythmicity, drive dEMG and NLP EMG activity.

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.

The splanchnic sympathetic nerves exert a powerful anti‐inflammatory action in response to systemic administration of lipopolysaccharide in rats (Martelli et al. J. Physiol. 592: 1677–1686, 2014). However, it is not known how this endogenous anti‐inflammatory influence of the splanchnic nerve affects the body’s ability to fight infection. The aim of this study was to determine if and how the endogenous reflex activation of the splanchnic anti‐inflammatory pathway affects blood cytokine levels and the disease course of bacteremia after intravenous infusion of live E. coli into a conscious large animal (sheep). One month prior to infection, the greater splanchnic nerve was cut bilaterally (SplancX, n = 8) or sham surgery (Sham, n = 7) performed in anesthetized sheep (isoflurane/O2). After 1 month, these conscious sheep were infused with live E. coli (2.8 × 109 CFU in 30 min) into a jugular vein. Blood samples were obtained at 1.5, 3, 6, 24 and 48 h after bacterial injection. As expected, i.v. infusion of E. coli caused systemic bacteremia with high blood counts of E. coli [3.02 ± 0.64 Log10(CFU/ml + 1), mean ± sem] in 5 of 7 sham sheep. By contrast, no significant bacteremia was observed in any of the 8 SplancX sheep (0.18 ± 0.18 Log10[CFU/ml + 1], p < 0.01, c.f. Sham sheep). Plasma levels of tumour necrosis factor‐α peaked after 1.5 h, and were significantly greater in SplancX vs Sham sheep (2.4 ± 0.6 vs 1.5 ± 0.2 ng/ml, p < 0.05) at this time. Plasma levels of another pro‐inflammatory cytokine, interleukin‐6, peaked at 6h and were higher in SplancX vs Sham sheep (7.4 ± 0.6 vs 2.4 ± 0.6 ng/ml, p < 0.01). Although plasma levels of the anti‐inflammatory cytokine interleukin‐10 increased in both groups, they were lower in SplancX vs Sham sheep from 1.5 h to 24 h (2.9 ± 0.4 vs 6.7 ± 1.1 ng/ml at 24 h, p < 0.01). All plasma cytokine levels measured had returned to baseline by 48 h. Plasma cortisol levels were higher (p<0.01) in SplancX sheep only at 6 h (298 ± 29 vs 138 ± 37 ng/ml), while the fever was less (p < 0.05) in SplancX at 6 h (40.3 ± 0.3 vs 40.8 ± 0.3°C). The greater pro‐inflammatory response in splanchnic denervated animals most likely contributes to their enhanced ability to clear bacteremia. The present findings demonstrate the powerful consequences of neural control of inflammation in the context of an acute systemic infection.Support or Funding InformationSupported by the National Heart Foundation of Australia

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.

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.

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. AbstractWe 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.

Norepinephrine exacerbates renal medullary hypoxia in experimental septic acute kidney injury. Here we examined whether dexmedetomidine, an alpha 2-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 pO(2) (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 mu g/kg/h) co-administration significantly reduced the norepinephrine dose (0.8 to 0.4 mu g/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.

The nervous and immune systems talk to and influence each other. In particular, the nervous system helps maintain inflammation within appropriate limits. It is known that a neural reflex, termed the inflammatory reflex, is engaged and inhibits inflammation in response to lipopolysaccharide, a component of the wall of gram negative bacteria. This reflex has its efferent motor arm in the splanchnic nerves. Viral infections are different from bacterial infections and the innate immune system detects and responds differently to them.The aim of this study was to test the hypothesis that the inflammatory reflex is involved in the control of inflammation triggered by viruses as well as bacteria.For this purpose, we investigated whether the injection of polyinosinic:polycytidylic acid (Poly I:C; 1mg/kg i.v.), which mimics viral challenge, is able to activate splanchnic sympathetic nerve activity (SSNA) in urethane anesthetized rats. We also studied the effect of bilateral section of the greater splanchnic nerves on the systemic inflammatory response 90 minutes after the injection of the same dose of Poly I:C.Our results showed that poly I:C induces an activation of the SSNA that lasted for at least 7 hours. Furthermore, splanchnic nerve section resulted respectively in 3‐fold higher and 4‐fold lower levels of the pro‐inflammatory cytokine plasma tumor necrosis factor α (TNF) and the anti‐inflammatory cytokine interleukin 10.These results show that a viral challenge activates the inflammatory reflex in the same way as a bacterial challenge. Viral infections can lead to secondary bacterial infections. Because viral stimulation of the inflammatory reflex would suppress innate immune function, this may be why the host's ability to fight off a secondary attack by bacteria is compromised.Support or Funding InformationThis work was supported by project grant number 1098887 from the National Health and Medical Research Council (NHMRC) of Australia and from the Victorian Government Operational Infrastructure Support Program. E. N. Komegae was the recipient of a postdoctoral fellowship from Fundação de Amparo a Pesquisa do Estado de São Paulo (FAPESP 2016/1555‐6).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Electrical stimulation of the vagus nerve (VNS) is a novel strategy used to treat inflammatory conditions. Therapeutic VNS activates both efferent and afferent fibers; however, the effects attributable to vagal afferent stimulation are unclear. Here, we tested if selective activation of afferent fibers in the abdominal vagus suppresses systemic inflammation. In urethane-anesthetized rats challenged with lipopolysaccharide (LPS, 60 mu g/kg, i.v.), abdominal afferent VNS (2 Hz for 20 min) reduced plasma tumor necrosis factor alpha (TNF) levels 90 min later by 88% compared with unmanipulated animals. Pre-cutting the cervical vagi blocked this anti-inflammatory action. Interestingly, the surgical procedure to expose and prepare the abdominal vagus for afferent stimulation ('vagal manipulation') also had an anti-inflammatory action. Levels of the anti-inflammatory cytokine IL-10 were inversely related to those of TNF. Prior bilateral section of the splanchnic sympathetic nerves reversed the anti-inflammatory actions of afferent VNS and vagal manipulation. Sympathetic efferent activity in the splanchnic nerve was shown to respond reflexly to abdominal vagal afferent stimulation. These data demonstrate that experimentally activating abdominal vagal afferent fibers suppresses systemic inflammation, and that the efferent neural pathway for this action is in the splanchnic sympathetic nerves.

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.

In response to a systemic inflammatory challenge, the CNS orchestrates several stereotyped responses. First, the hypothalamic‐pituitary‐adrenal axis is activated. Second, there is fever. Third, the splanchnic anti‐inflammatory pathway (SAIP), the efferent arm of the inflammatory reflex, is activated to inhibit an excessive release of pro‐inflammatory markers (Martelli et al. Exp Physiol 2016;101(10):1245–1252). The first two responses, dependent on prostaglandin synthesis, are blocked by cyclooxygenase (COX) inhibitors (Garcia‐Bueno et al. J Neurosci 2009;29(41):12970–81; Steiner et al. Am J Phys Regul Integr Comp Physiol 2009;297(2):R485–94). We report here that the SAIP is not. In rats, bilateral section of the splanchnic nerves enhanced the plasma levels of TNF‐α 90 mins after i.v. lipopolysaccharide (LPS; 60 μg/kg) several‐fold, and this remained the case in animals treated with the COX inhibitor diclofenac (5mg/kg × 2 injections i.v., before and 45 minutes after LPS injection), even though fever was blocked. Activation of the SAIP by LPS was evidently unaffected by COX inhibition sufficient to block fever. Using this knowledge, we set out to identify which sympathetic premotor neurons drive the SAIP, removing the confound of fever. In preparatory surgery, the retrograde tracer Fast Blue was injected around the IML of the lower thoracic cord in 18 rats. Two weeks later, the femoral vein was cannulated and the animal allowed to recover for 48 hours. Diclofenac (5mg/kg) was given i.v. 10 mins before LPS (60 μg/kg, i.v.). 150 mins later rats were deeply anesthetized and perfusion‐fixed. Brains were frozen sectioned and processed immunohistochemically for Fos. Double fluorescence microscopy revealed fast blue labeled (spinally –projecting) and Fos‐labeled neurons. The only site found to show double labeled neurons was a sub‐area of the hypothalamic paraventricular nucleus. These may be the premotor neurons that drive the SAIP. Support or Funding Information DM is supported by NHMRC (GNT1098887) and an IBRO award. STY is supported by grants from the NHMRC (GNT1079680). The Florey Institute of Neuroscience and Mental Health acknowledges the strong support from the Victorian Government and in particular the funding from the Operational Infrastructure Support Grant

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. AbstractCardiac 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.