Prodromal Parkinson disease signs are predicted by a whole-blood inflammatory transcriptional signature in young Pink1-/- rats.

BACKGROUND: Parkinson disease (PD) is the fastest growing neurodegenerative disease. The molecular pathology of PD in the prodromal phase is poorly understood; as such, there are no specific prognostic or diagnostic tests. A validated Pink1 genetic knockout rat was used to model early-onset and progressive PD. Male Pink1-/- rats exhibit progressive declines in ultrasonic vocalizations as well as hindlimb and forelimb motor deficits by mid-to-late adulthood. Previous RNA-sequencing work identified upregulation of genes involved in disease pathways and inflammation within the brainstem and vocal fold muscle. The purpose of this study was to identify gene pathways within the whole blood of young Pink1-/- rats (3 months of age) and to link gene expression to early acoustical changes. To accomplish this, limb motor testing (open field and cylinder tests) and ultrasonic vocalization data were collected, immediately followed by the collection of whole blood and RNA extraction. Illumina® Total RNA-Seq TruSeq platform was used to profile differential expression of genes. Statistically significant genes were identified and Weighted Gene Co-expression Network Analysis was used to construct co-expression networks and modules from the whole blood gene expression dataset as well as the open field, cylinder, and USV acoustical dataset. ENRICHR was used to identify the top up-regulated biological pathways. RESULTS: The data suggest that inflammation and interferon signaling upregulation in the whole blood is present during early PD. We also identified genes involved in the dysregulation of ribosomal protein and RNA processing gene expression as well as prion protein gene expression. CONCLUSIONS: These data identified several potential blood biomarkers and pathways that may be linked to anxiety and vocalization acoustic parameters and are key candidates for future drug-repurposing work and comparison to human datasets.

Assessment of Efficacy and Safety of Zagotenemab: Results From PERISCOPE-ALZ, a Phase 2 Study in Early Symptomatic Alzheimer Disease.

BACKGROUND AND OBJECTIVES: Zagotenemab (LY3303560), a monoclonal antibody that preferentially targets misfolded, extracellular, aggregated tau, was assessed in the PERISCOPE-ALZ phase 2 study to determine its ability to slow cognitive and functional decline relative to placebo in early symptomatic Alzheimer disease (AD). METHODS: Participants were enrolled across 56 sites in North America and Japan. Key eligibility criteria included age of 60-85 years, Mini-Mental State Examination score of 20-28, and intermediate levels of brain tau on PET imaging. In this double-blind study, participants were equally randomized to 1,400 mg or 5,600 mg of zagotenemab, or placebo (IV infusion every 4 weeks for 100 weeks). The primary outcome was change on the Integrated AD Rating Scale (iADRS) assessed by a Bayesian Disease Progression model. Secondary measures include mixed model repeated measures analysis of additional cognitive and functional endpoints as well as biomarkers of AD pathology. RESULTS: A total of 360 participants (mean age = 75.4 years; female = 52.8%) were randomized, and 218 completed the treatment period. Demographics and baseline characteristics were reasonably balanced among arms. The mean disease progression ratio (proportional decline in the treated vs placebo group) with 95% credible intervals for the iADRS was 1.10 (0.959-1.265) for the zagotenemab low-dose group and 1.05 (0.907-1.209) for the high-dose, where a ratio less than 1 favors the treatment group. Secondary clinical endpoint measures failed to show a drug-placebo difference in favor of zagotenemab. No treatment effect was demonstrated by flortaucipir PET, volumetric MRI, or neurofilament light chain (NfL) analyses. A dose-related increase in plasma phosphorylated tau181 and total tau was demonstrated. Zagotenemab treatment groups reported a higher incidence of adverse events (AEs) (85.1%) compared with the placebo group (74.6%). This difference was not attributable to any specific AE or category of AEs. DISCUSSION: In participants with early symptomatic AD, zagotenemab failed to achieve significant slowing of clinical disease progression compared with placebo. Imaging biomarker and plasma NfL findings did not show evidence of pharmacodynamic activity or disease modification. TRIAL REGISTRATION INFORMATION: ClinicalTrials.gov: NCT03518073. CLASSIFICATION OF EVIDENCE: This study provides Class II evidence that for patients with early symptomatic AD, zagotenemab does not slow clinical disease progression.

Safety, Tolerability, and Pharmacokinetics of Zagotenemab in Participants with Symptomatic Alzheimer's Disease: A Phase I Clinical Trial.

Background: Zagotenemab (LY3303560), a monoclonal antibody, preferentially binds to extracellular, misfolded, aggregated tau that has been implicated in Alzheimer's disease (AD). Objective: The goal of this study was to assess the safety and pharmacokinetics of multiple doses of zagotenemab in participants with AD. Methods: This was a Phase Ib, multi-site, participant- and investigator-blind, placebo-controlled, parallel-group study in participants with mild cognitive impairment due to AD or mild to moderate AD. After screening, participants were randomized to zagotenemab 70 mg, 210 mg, or placebo every 4 weeks for up to 49 weeks and were followed up for 16 weeks. Results: A total of 13 males and 9 females, aged 59 to 84 years, were dosed. No deaths occurred during this study. A total of 4 serious adverse events occurred in 2 participants who then discontinued the study. The most commonly reported (3 or more participants) treatment-emergent adverse events were sinus bradycardia, headache, fall, and bronchitis. The pharmacokinetics profile showed generally linear exposures across the dose range studied with a clearance of ~8 mL/h. The half-life of zagotenemab in serum was ~20 days. A dose-dependent increase in plasma tau was observed. No other significant pharmacodynamic differences were observed due to low dose levels and limited treatment duration. Conclusions: No dose-limiting adverse events were observed with zagotenemab treatment. Pharmacokinetics of zagotenemab were typical for a monoclonal antibody. Meaningful pharmacodynamic differences were not observed.Clinicaltrials.gov: NCT03019536.

Prodromal Parkinson disease signs are predicted by a whole-blood inflammatory transcriptional signature in young Pink1-/- rats.

Background: Parkinson disease (PD) is the fastest growing neurodegenerative disease. The molecular pathology of PD in the prodromal phase is poorly understood; as such, there are no specific prognostic or diagnostic tests. A validated Pink1 genetic knockout rat was used to model early-onset and progressive PD. Male Pink1-/- rats exhibit progressive declines in ultrasonic vocalizations as well as hindlimb and forelimb motor deficits by mid-to-late adulthood. Previous RNA-sequencing work identified upregulation of genes involved in disease pathways and inflammation within the brainstem and vocal fold muscle. The purpose of this study was to identify gene pathways within the whole blood of young Pink1-/- rats (3 months of age) and to link gene expression to early acoustical changes. To accomplish this, limb motor testing (open field and cylinder tests) and ultrasonic vocalization data were collected, immediately followed by the collection of whole blood and RNA extraction. Illumina® Total RNA-Seq TruSeq platform was used to profile differential expression of genes. Statistically significant genes were identified and Weighted Gene Co-expression Network Analysis was used to construct co-expression networks and modules from the whole blood gene expression dataset as well as the open field, cylinder, and USV acoustical dataset. ENRICHR was used to identify the top up-regulated biological pathways. Results: The data suggest that inflammation and interferon signaling upregulation in the whole blood is present during early PD. We also identified genes involved in the dysregulation of ribosomal protein and RNA processing gene expression as well as prion protein gene expression. Conclusions: These data identified several potential blood biomarkers and pathways that may be linked to anxiety and vocalization acoustic parameters and are key candidates for future drug-repurposing work and comparison to human datasets.

Thyroarytenoid Oxidative Metabolism and Synaptic Signaling Dysregulation in the Female Pink1-/- Rat.

OBJECTIVES AND HYPOTHESIS: Vocal dysfunction, including hypophonia, in Parkinson disease (PD) manifests in the prodromal period and significantly impacts an individual's quality of life. Data from human studies suggest that pathology leading to vocal deficits may be structurally related to the larynx and its function. The Pink1-/- rat is a translational model used to study pathogenesis in the context of early-stage mitochondrial dysfunction. The primary objective of this work was to identify differentially expressed genes in the thyroarytenoid muscle and examine the dysregulated biological pathways in the female rat. METHODS: RNA sequencing was used to determine thyroarytenoid (TA) muscle gene expression in adult female Pink1-/- rats compared with controls. A bioinformatic approach and the ENRICHR gene analysis tool were used to compare the sequencing dataset with biological pathways and processes, disease relationships, and drug-repurposing compounds. Weighted Gene Co-expression Network Analysis was used to construct biological network modules. The data were compared with a previously published dataset in male rats. RESULTS: Significant upregulated pathways in female Pink1-/- rats included fatty acid oxidation and muscle contraction, synaptic transmission, and neuromuscular processes. Downregulated pathways included anterograde transsynaptic signaling, chemical synaptic transmission, and ion release. Several drug treatment options including cetuximab, fluoxetine, and resveratrol are hypothesized to reverse observed genetic dysregulation. CONCLUSIONS: Data presented here are useful for identifying biological pathways that may underlie the mechanisms of peripheral dysfunction including neuromuscular synaptic transmission to the TA muscle. These experimental biomarkers have the potential to be targeted as sites for improving the treatment for hypophonia in early-stage PD. LEVEL OF EVIDENCE: NA Laryngoscope, 133:3412-3421, 2023.

Early ultrasonic vocalization deficits and related thyroarytenoid muscle pathology in the transgenic TgF344-AD rat model of Alzheimer's disease.

Background: Alzheimer's disease (AD) is a progressive neurologic disease and the most common cause of dementia. Classic pathology in AD is characterized by inflammation, abnormal presence of tau protein, and aggregation of ß-amyloid that disrupt normal neuronal function and lead to cell death. Deficits in communication also occur during disease progression and significantly reduce health, well-being, and quality of life. Because clinical diagnosis occurs in the mid-stage of the disease, characterizing the prodrome and early stages in humans is currently challenging. To overcome these challenges, we use the validated TgF344-AD (F344-Tg(Prp-APP, Prp-PS1)19/Rrrc) transgenic rat model that manifests cognitive, behavioral, and neuropathological dysfunction akin to AD in humans. Objectives: The overarching goal of our work is to test the central hypothesis that pathology and related behavioral deficits such as communication dysfunction in part manifest in the peripheral nervous system and corresponding target tissues already in the early stages. The primary aims of this study are to test the hypotheses that: (1) changes in ultrasonic vocalizations (USV) occur in the prodromal stage at 6 months of age and worsen at 9 months of age, (2) inflammation as well as AD-related pathology can be found in the thyroarytenoid muscle (TA) at 12 months of age (experimental endpoint tissue harvest), and to (3) demonstrate that the TgF344-AD rat model is an appropriate model for preclinical investigations of early AD-related vocal deficits. Methods: USVs were collected from male TgF344-AD (N = 19) and wildtype (WT) Fischer-344 rats (N = 19) at 6 months (N = 38; WT: n = 19; TgF344-AD: n = 19) and 9 months of age (N = 18; WT: n = 10; TgF344-AD: n = 8) and acoustically analyzed for duration, mean power, principal frequency, low frequency, high frequency, peak frequency, and call type. RT-qPCR was used to assay peripheral inflammation and AD-related pathology via gene expressions in the TA muscle of male TgF344-AD rats (n = 6) and WT rats (n = 6) at 12 months of age. Results: This study revealed a significant reduction in mean power of ultrasonic calls from 6 to 9 months of age and increased peak frequency levels over time in TgF344-AD rats compared to WT controls. Additionally, significant downregulation of AD-related genes Uqcrc2, Bace2, Serpina3n, and Igf2, as well as downregulation of pro-inflammatory gene Myd88 was found in the TA muscle of TgF344-AD rats at 12 months of age. Discussion: Our findings demonstrate early and progressive vocal deficits in the TgF344-AD rat model. We further provide evidence of dysregulation of AD-pathology-related genes as well as inflammatory genes in the TA muscles of TgF344-AD rats in the early stage of the disease, confirming this rat model for early-stage investigations of voice deficits and related pathology.

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.