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The nodose and jugular vagal ganglia supply sensory innervation to the airways and lungs. Jugular vagal airway sensory neurons wire into a brainstem circuit with ascending projections into the submedius thalamic nucleus (SubM) and ventrolateral orbital cortex (VLO), regions known to regulate the endogenous analgesia system. Here we investigate whether the SubM–VLO circuit exerts descending regulation over airway vagal reflexes in male and female rats using a range of neuroanatomical tracing, reflex physiology, and chemogenetic techniques. Anterograde and retrograde neuroanatomical tracing confirmed the connectivity of the SubM and VLO. Laryngeal stimulation in anesthetized rats reduced respiration, a reflex that was potently inhibited by activation of SubM. Conversely, inhibition of SubM potentiated laryngeal reflex responses, while prior lesions of VLO abolished the effects of SubM stimulation. In conscious rats, selective chemogenetic activation of SubM neurons specifically projecting to VLO significantly inhibited respiratory responses evoked by inhalation of the nociceptor stimulant capsaicin. Jugular vagal inputs to SubM via the medullary paratrigeminal nucleus were confirmed using anterograde transsynaptic conditional herpes viral tracing. Respiratory responses evoked by microinjections of capsaicin into the paratrigeminal nucleus were significantly attenuated by SubM stimulation, whereas those evoked via the nucleus of the solitary tract were unaltered. These data suggest that jugular vagal sensory pathways input to a nociceptive thalamocortical circuit capable of regulating jugular sensory processing in the medulla. This circuit organization suggests an intersection between vagal sensory pathways and the endogenous analgesia system, potentially important for understanding vagal sensory processing in health and mechanisms of hypersensitivity in disease.SIGNIFICANCE STATEMENTJugular vagal sensory pathways are increasingly recognized for their important role in defensive respiratory responses evoked from the airways. Jugular ganglia neurons wire into a central circuit that is notable for overlapping with somatosensory processing networks in the brain rather than the viscerosensory circuits in receipt of inputs from the nodose vagal ganglia. Here we demonstrate a novel and functionally relevant example of intersection between vagal and somatosensory processing in the brain. The findings of the study offer new insights into interactions between vagal and spinal sensory processing, including the medullary targets of the endogenous analgesia system, and offer new insights into the central processes involved in airway defense in health and disease.

Key points Airway projecting sensory neurons arising from the jugular vagal ganglia terminate centrally in the brainstem paratrigeminal nucleus, synapsing upon neurons expressing the neurokinin 1 receptor. This study aimed to assess the involvement of paratrigeminal neurokinin 1 receptor neurons in the regulation of cough, breathing and airway defensive responses. Lesioning neurokinin 1 receptor expressing paratrigeminal neurons significantly reduced cough evoked by inhaled bradykinin but not inhaled ATP or tracheal mechanical stimulation. The reduction in bradykinin-evoked cough was not accompanied by changes in baseline or evoked respiratory variables (e.g. frequency, volume or timing), animal avoidance behaviours or the laryngeal apnoea reflex. These findings warrant further investigations into targeting the jugular ganglia and paratrigeminal nucleus as a therapy for treating cough in disease. Abstract Jugular vagal ganglia sensory neurons innervate the large airways and are thought to mediate cough and associated perceptions of airway irritations to a range of chemical irritants. The central terminals of jugular sensory neurons lie within the brainstem paratrigeminal nucleus, where postsynaptic neurons can be differentiated based on the absence or presence of the neurokinin 1 (NK1) receptor. Therefore, in the present study, we set out to test the hypothesis that NK1 receptor expressing paratrigeminal neurons play a role in cough evoked by inhaled chemical irritants. To test this, we performed selective neurotoxin lesions of NK1 receptor expressing neurons in the paratrigeminal nucleus in guinea-pigs using substance P conjugated to saporin (SSP-SAP). Sham lesion control or SSP-SAP lesion guinea-pigs received nebulised challenges, with the pan-nociceptor stimulant bradykinin or the nodose ganglia specific stimulant adenosine 5'-triphosphate (ATP), in conscious whole-body plethysmography to study cough and associated behaviours. Laryngeal apnoea reflexes and cough evoked by mechanical stimulation of the trachea were additionally investigated in anaesthetised guinea-pigs. SSP-SAP significantly and selectively reduced the number of NK1 receptor expressing neurons in the paratrigeminal nucleus. This was associated with a significant reduction in bradykinin-evoked cough, but not ATP-evoked cough, mechanical cough or laryngeal apnoeic responses. These data provide further evidence for a role of jugular vagal pathways in cough, and additionally suggest an involvement of NK1 receptor expressing neurons in the paratrigeminal nucleus. Therefore, this neural pathway may provide novel therapeutic opportunities to treat conditions of chronic cough.

A learning mechanism in the striatum An intriguing characteristic of the striatum is the random spatial distribution and high degree of intermingling between expression of dopamine receptor types 1 (D1) and 2 (D2) within striatal projection neurons (SPNs). The resulting highly entropic mosaic extends through a homogeneous space and is mostly devoid of histological boundaries. The rules established locally by D1- and D2-expressing SPNs (D1-SPNs and D2-SPNs) are thus likely critical in defining how functional territories develop throughout the striatum. Matamales et al. found that activated D2-SPNs access and modify developing behavioral programs encoded by regionally defined ensembles of transcriptionally active D1-SPNs. This process is slow because it depends on the molecular integration of additive neuro-modulatory signals. However, with time, it creates the regional functional boundaries that are necessary to identify and shape specific learning in the striatum. Science , this issue p. 549

Vagal sensory neurons contribute to the symptoms and pathogenesis of inflammatory pulmonary diseases through processes that involve changes to their morphological and functional characteristics. The alarmin high mobility group box-1 (HMGB1) is an early mediator of pulmonary inflammation and can have actions on neurons in a range of inflammatory settings. We hypothesized that HMGB1 can regulate the growth and function of vagal sensory neurons and we set out to investigate this and the mechanisms involved. Culturing primary vagal sensory neurons from wildtype mice in the presence of HMGB1 significantly increased neurite outgrowth, while acute application of HMGB1 to isolated neurons under patch clamp electrophysiological investigation produced inward currents and enhanced action potential firing. Transcriptional analyses revealed the expression of the cognate HMGB1 receptors, Receptor for Advanced Glycation End products (RAGE) and Toll-like Receptor 4 (TLR4), in subsets of vagal sensory neurons. HMGB1-evoked growth and electrophysiological responses were significantly reduced in primary vagal sensory neurons harvested from RAGE deficient mice and completely absent in neurons from RAGE/TLR4 double deficient mice. Immunohistochemical analysis of vagal sensory neurons collected from mice after intranasal infection with murine pneumovirus or influenza A virus (IAV), or after intratracheal administration with the viral mimetic PolyI:C, revealed a significant increase in nuclear-to-cytoplasm translocation of HMGB1 compared to mock-inoculated mice. Neurons cultured from virus infected wildtype mice displayed a significant increase in neurite outgrowth, which was not observed for neurons from virus infected RAGE or RAGE/TLR4 deficient mice. These data suggest that HMGB1 can enhance vagal sensory neuron growth and excitability, acting primarilyviasensory neuron RAGE. Activation of the HMGB1-RAGE axis in vagal sensory neurons could be an important mechanism leading to vagal hyperinnervation and hypersensitivity in chronic pulmonary disease.

Bronchopulmonary sensory neurons are derived from the vagal sensory ganglia and are essential for monitoring the physical and chemical environment of the airways and lungs. Subtypes are heterogenous in their responsiveness to stimuli, phenotype, and developmental origin, but they collectively serve to regulate normal respiratory and pulmonary processes and elicit a diverse range of defensive physiological responses that protect against noxious stimuli. In this study, we aimed to investigate the transcriptional features of vagal bronchopulmonary sensory neurons using single-cell RNA sequencing (scRNA-seq) to provide a deeper insight into their molecular profiles. Retrogradely labeled vagal sensory neurons projecting to the airways and lungs were hierarchically clustered into five types reflecting their developmental lineage (neural crest versus placodal) and putative function (nociceptors versus mechanoreceptors). The purinergic receptor subunit P2rx2 is known to display restricted expression in placodal-derived nodose neurons, and we demonstrate that the gene profiles defining cells high and low in expression of P2rx2 include G protein coupled receptors and ion channels, indicative of preferential expression in nodose or jugular neurons. Our results provide valuable insight into the transcriptional characteristics of bronchopulmonary sensory neurons and provide rational targets for future physiological investigations.

Chronic cough is a difficult to treat symptom of many respiratory and some non-respiratory diseases, indicating that varied pathologies can underpin the development of chronic cough. However, clinically and experimentally it has been useful to collate these different pathological processes into the single unifying concept of cough hypersensitivity. Cough hypersensitivity syndrome is reflected by troublesome cough often precipitated by levels of stimuli that ordinarily don't cause cough in healthy people, and this appears to be a hallmark feature in many patients with chronic cough. Accordingly, a strong argument has emerged that changes in the excitability and/or normal regulation of the peripheral and central neural circuits responsible for cough are instrumental in establishing cough hypersensitivity and for causing excessive cough in disease. In this review, we explore the current peripheral and central neural mechanisms that are believed to be involved in altered cough sensitivity and present possible links to the mechanism of action of novel therapies that are currently undergoing clinical trials for chronic cough.

Influenza A virus (IAV) is rapidly detected in the airways by the immune system, with resident parenchymal cells and leukocytes orchestrating viral sensing and the induction of antiviral inflammatory responses. The airways are innervated by heterogeneous populations of vagal sensory neurons which also play an important role in pulmonary defense. How these neurons respond to IAV respiratory infection remains unclear. Here, we use a murine model to provide the first evidence that vagal sensory neurons undergo significant transcriptional changes following a respiratory IAV infection. RNA sequencing on vagal sensory ganglia showed that IAV infection induced the expression of many genes associated with an antiviral and pro-inflammatory response and this was accompanied by a significant increase in inflammatory cell recruitment into the vagal ganglia. Assessment of gene expression in single-vagal sensory neurons confirmed that IAV infection induced a neuronal inflammatory phenotype, which was most prominent in bronchopulmonary neurons, and also evident in some neurons innervating other organs. The altered transcriptome could be mimicked by intranasal treatment with cytokines and the lung homogenates of infected mice, in the absence of infectious virus. These data argue that IAV pulmonary infection and subsequent inflammation induces vagal sensory ganglia neuroinflammation and this may have important implications for IAV-induced morbidity.

Cholinergic basal forebrain (cBF)-derived neurotransmission plays a crucial role in regulating neuronal function throughout the cortex, yet the mechanisms controlling cholinergic innervation to downstream targets have not been elucidated. Here we report that removing the p75 neurotrophin receptor (p75NTR) from cBF neurons induces a significant impairment in fear extinction consolidation. We demonstrate that this is achieved through alterations in synaptic connectivity and functional activity within the medial prefrontal cortex. These deficits revert back to wild-type levels upon re-expression of the active domain of p75NTR in adult animals. These findings demonstrate a novel role for cholinergic neurons in fear extinction consolidation and suggest that neurotrophic signaling is a key regulator of cholinergic-cortical innervation and function.

Sensory information arising from the airways is processed in a distributed brain network that encodes for the discriminative and affective components of the resultant sensations. These higher brain networks in turn regulate descending motor control circuits that can both promote or suppress behavioural responses. Here we explore the existence of possible descending neural control pathways that regulate airway afferent processing in the brainstem, analogous to the endogenous descending analgesia system described for noxious somatosensation processing and placebo analgesia. A key component of this circuitry is the midbrain periaqueductal grey, a region of the brainstem recently highlighted for its altered activity in patients with chronic cough. Understanding the nature and plasticity of descending neural control may help identify novel central therapeutic targets to alleviate the neuronal hypersensitivity underpinning many symptoms of respiratory disease.

Extinction learning allows animals to withhold voluntary actions that are no longer related to reward and so provides a major source of behavioral control. Although such learning is thought to depend on dopamine signals in the striatum, the way the circuits mediating goal-directed control are reorganized during new learning remains unknown. Here, by mapping a dopamine-dependent transcriptional activation marker in large ensembles of striatal projection neurons (SPNs) expressing dopamine receptor type 1 (D1-SPNs) or 2 (D2-SPNs) in mice, we demonstrate an extensive and dynamic D2- to D1-SPN trans-modulation across the dorsal striatum that is necessary for updating previous goal-directed learning. Our findings suggest that D2-SPNs suppress the influence of outdated D1-SPN plasticity within functionally relevant striatal territories to reshape volitional action.

The airways receive a dense supply of sensory nerve fibers that are responsive to damaging or potentially injurious stimuli. These airway nociceptors are mainly derived from the jugular and nodose vagal ganglia, and when activated they induce a range of reflexes and sensations that play an essential role in airway protection. Jugular nociceptors differ from nodose nociceptors in their embryonic origins, molecular profile and termination patterns in the airways and the brain, and recent discoveries suggest that excessive activity in jugular nociceptors may be central to the development of chronic cough. For these reasons, targeting jugular airway nociceptor signaling processes at different levels of the neuraxis may be a promising target for therapeutic development. In this focused review, we present the current understanding of jugular ganglia nociceptors, how they may contribute to chronic cough and mechanisms that could be targeted to bring about cough suppression.

Airway afferents monitor the local chemical and physical micro-environments in the airway wall and lungs and send this information centrally to regulate neural circuits involved in setting autonomic tone, evoking reflex and volitional respiratory motor outflows, encoding perceivable sensations and contributing to higher order cognitive processing. In this mini-review we present a current overview of the central wiring of airway afferent circuits in the brainstem and brain, highlighting recent discoveries that augment our understanding of airway sensory processing. We additionally explore how advances in describing the molecular diversity of airway afferents may influence future research efforts aimed at defining central mesoscale connectivity of airway afferent pathways. A refined understanding of how functionally distinct airway afferent pathways are organized in the brain will provide deeper insight into the physiology of airway afferent-evoked responses and may foster opportunities for targeted modulation of specific pathways involved in disease.

The respiratory system is densely innervated by sensory neurons arising from the jugular (superior) and nodose (inferior) vagal ganglia. However, a distinction exists between jugular and nodose neurons as these ganglia developmentally originate from the neural crest and the epibranchial placodes, respectively. This different embryological origin underpins an important source of heterogeneity in vagal afferent biology, and may extend to include fundamentally different central neural circuits that are in receipt of jugular versus nodose afferent inputs. Indeed, recent studies using viral tract tracing and human brain imaging support the notion that airway sensors contribute inputs to multiple central circuits. Understanding the neural pathways arising from the airways and lungs may provide novel insights into aberrant sensations, such as the urge-to-cough, characteristic of respiratory disease.

Cough is an essential defensive behavior for maintaining airway patency and to protect the lungs from potentially harmful agents. However, inflammatory pathologies can sensitize and activate the neural pathways regulating cough, leading to excessive and nonproductive coughing that serves little protective utility. Problematic cough continues to be one of the most common reasons for seeking medical advice, yet for many patients, it can be refractory to disease-specific treatments and currently available antitussive therapies. The effect of inflammation on cough neural processing occurs not only at the level of the bronchopulmonary sensory nerve terminals but also within the nervous system at multiple peripheral and central sites. Sensory nerves also actively regulate inflammation, and it is therefore a complex interplay between the immune and nervous systems that contributes to chronic cough and the associated sensory hypersensitivities. In this review we provide a brief overview of cough neurobiology in health and disease and then explore the peripheral and central nervous system sites at which neuroimmune interactions can occur. We present advancements in the development of effective antitussive therapies and suggest novel targets for future consideration.

Sensory neurons of the jugular vagal ganglia innervate the respiratory tract and project to the poorly studied medullary paratrigeminal nucleus. In the present study, we used neuroanatomical tracing, pharmacology and physiology in guinea pig to investigate the paratrigeminal neural circuits mediating jugular ganglia-evoked respiratory reflexes. Retrogradely traced laryngeal jugular ganglia neurons were largely (> 60%) unmyelinated and expressed the neuropeptide substance P and calcitonin gene-related peptide, although a population (~ 30%) of larger diameter myelinated jugular neurons was defined by the expression of vGlut1. Within the brainstem, vagal afferent terminals were confined to the caudal two-thirds of the paratrigeminal nucleus. Electrical stimulation of the laryngeal mucosa evoked a vagally mediated respiratory slowing that was mimicked by laryngeal capsaicin application. These laryngeal reflexes were modestly reduced by neuropeptide receptor antagonist microinjections into the paratrigeminal nucleus, but abolished by ionotropic glutamate receptor antagonists. D,L-Homocysteic acid microinjections into the paratrigeminal nucleus mimicked the laryngeal-evoked respiratory slowing, whereas capsaicin microinjections evoked a persistent tachypnoea that was insensitive to glutamatergic inhibition but abolished by neuropeptide receptor antagonists. Extensive projections from paratrigeminal neurons were anterogradely traced throughout the pontomedullary respiratory column. Dual retrograde tracing from pontine and ventrolateral medullary termination sites, as well as immunohistochemical staining for calbindin and neurokinin 1 receptors, supported the existence of different subpopulations of paratrigeminal neurons. Collectively, these data provide anatomical and functional evidence for at least two types of post-synaptic paratrigeminal neurons involved in respiratory reflexes, highlighting an unrecognised complexity in sensory processing in this region of the brainstem.

Cough and the accompanying sensation known as the urge-to-cough are complex neurobiological phenomena dependent on sensory and motor neural processing at many levels of the neuraxis. In addition to the excitatory neural circuits that provide the positive drive for inducing cough and the urge-to-cough, recent studies have highlighted the existence of likely inhibitory central neural processes that can be engaged to suppress cough sensorimotor processing. In many respects, the balance between excitatory and inhibitory central cough control may be a critical determinant of cough in health and disease which argues for the importance of understanding the biology of these putative central inhibitory processes. This brief review summarises the current knowledge of the central circuits that govern voluntary and involuntary cough suppression and posits the notion of targeting central suppressive mechanisms as a treatment for disordered cough in disease.

Changes in cardiorespiratory control accompany the expression of complex emotions, indicative of limbic brain inputs onto bulbar autonomic pathways. Previous studies have focussed on the role of the prefrontal cortex in autonomic regulation. However, the role of the hippocampus, also important in limbic processing, has not been addressed in detail. Anaesthetised, instrumented rats were used to map the location of hippocampal sites capable of evoking changes in cardiorespiratory control showing that stimulation of discrete regions within the CA1 fields of both the dorsal and ventral hippocampus potently alter breathing and cardiovascular activity. Additionally, tracing of the neuroanatomical tracts and pharmacological inactivation studies were used to demonstrate a role of the basomedial amygdala in hippocampal evoked responses. Collectively, these data support the existence of a hippocampal-amygdala neural circuit capable of modulating bulbar cardiorespiratory control networks and may suggest a role for this circuit in the top-down regulation of breathing and autonomic outflow necessary for the expression of complex emotions.

The airway sensory nervous system is composed of two anatomically distinct processing pathways that allow for the production of respiratory reflexes and voluntary evoked respiratory behaviours in response to sensing an airway irritation. Disordered sensory processing is a hallmark feature of many pulmonary disorders and results in the development of cough hypersensitivity syndrome, characterised by chronic cough and a persistent urge-to-cough in affected individuals. However, the mechanism underpinning how the airway sensory circuits become disordered, especially at the level of the central nervous system, is not well understood. In this mini-review we present well-defined mechanisms that lead to the development of chronic pain as a framework to explore the evidence that cough disorders may manifest due to neuroplasticity and sensitisation of important components of the airway sensory circuitry in the brain. We highlight recent discoveries of how airway sensory processing occurs in the brain in health and disease and additionally suggest areas where gaps exist in our current knowledge on the topic, with the goal of providing a better understanding of how airway circuits become dysfunctional in disease. This may in turn help identify novel therapeutic targets for restoring normal airway sensory processing and alleviating excessive cough.

Background and Purpose Subtypes of the hyperpolarization-activated cyclic nucleotide-gated (HCN) family of cation channels are widely expressed on nerves and smooth muscle cells in many organ systems, where they serve to regulate membrane excitability. Here we have assessed whether HCN channel inhibitors alter the function of airway smooth muscle or the neurons that regulate airway smooth muscle tone. Experimental Approach The effects of the HCN channel inhibitors ZD7288, zatebradine and Cs+ were assessed on agonist and nerve stimulation-evoked changes in guinea pig airway smooth muscle tone using tracheal strips in vitro, an innervated tracheal tube preparation ex vivo or in anaesthetized mechanically ventilated guinea pigs in vivo. HCN channel expression in airway nerves was assessed using immunohistochemistry, PCR and in situ hybridization. Key Results HCN channel inhibition did not alter airway smooth muscle reactivity in vitro to exogenously administered smooth muscle spasmogens, but significantly potentiated smooth muscle contraction evoked by the sensory nerve stimulant capsaicin and electrical field stimulation of parasympathetic cholinergic postganglionic neurons. Sensory nerve hyperresponsiveness was also evident in in vivo following HCN channel blockade. Cs+, but not ZD7288, potentiated preganglionic nerve-dependent airway contractions and over time induced autorhythmic preganglionic nerve activity, which was not mimicked by inhibitors of potassium channels. HCN channel expression was most evident in vagal sensory ganglia and airway nerve fibres. Conclusions and Implications HCN channel inhibitors had a previously unrecognized effect on the neural regulation of airway smooth muscle tone, which may have implications for some patients receiving HCN channel inhibitors for therapeutic purposes.

Many pulmonary diseases are characterized by inflammatory pathologies which in turn are responsible for obstruction, mucus hypersecretion, dyspnea, cough and other clinical symptoms of lung disease. Understanding processes that regulate inflammation will therefore provide insights into mechanisms that contribute to pulmonary dysfunction. The airways and lungs are densely innervated by autonomic and sensory nerves which might regulate aspects of pulmonary inflammation. In this review we provide a critical appraisal of the available literature on the topic of neuro-immune interactions in the airways and ask the question 'how strong is the evidence that pulmonary nerves regulate inflammation?'

Insights into the anatomical organization of complex neural circuits provide important information about function, and thus tools that facilitate neuroanatomical studies have proved invaluable in neuroscience. Advances in molecular cloning have allowed the production of novel recombinant neuroinvasive viruses for use in transynaptic neural tracing studies. However, the vast majority of these viruses have motility in the retrograde direction only, therefore limiting their use to studies of synaptic input circuitry. Here we describe the construction of an EGFP reporting herpes simplex virus, strain H129, which preferentially moves along synaptically connected neurons in the anterograde direction. In vitro and in vivo characterization studies confirm that the HSV-1 H129-EGFP retains comparable replication and neuroinvasiveness as the wildtype H129 virus. As a proof of principle we confirm anterograde movement of the H129-EGFP along polysynaptic pathways by inoculating the upper airways and tracking time-dependent EGFP expression in previously described ascending sensory pathways. These data confirm a genomic locus for recombining HSV-1 H129 such that normal viral function and replication is maintained. Novel viral recombinants such as HSV-1 H129-EGFP will be useful tools for delineating the central organization of peripheral sensory pathways as well as the synaptic outputs from central neuronal populations.

Sensory input from the airways to suprapontine brain regions contributes to respiratory sensations and the regulation of respiratory function. However, relatively little is known about the central organization of this higher brain circuitry. We exploited the properties of the H129 strain of herpes simplex virus 1 (HSV-1) to perform anterograde transneuronal tracing of the central projections of airway afferent nerve pathways. The extrathoracic trachea in Sprague-Dawley rats was inoculated with HSV-1 H129, and tissues along the neuraxis were processed for HSV-1 immunoreactivity. H129 infection appeared in the vagal sensory ganglia within 24 h and the number of infected cells peaked at 72 h. Brainstem nuclei, including the nucleus of the solitary tract and trigeminal sensory nuclei were infected within 48 h, and within 96 h infected cells were evident within the pons (lateral and medial parabrachial nuclei), thalamus (ventral posteromedial, ventral posterolateral, submedius, and reticular nuclei), hypothalamus (paraventricular and lateral nuclei), subthalamus (zona incerta), and amygdala (central and anterior amygdala area). At later times H129 was detected in cortical forebrain regions including the insular, orbital, cingulate, and somatosensory cortices. Vagotomy significantly reduced the number of infected cells within vagal sensory nuclei in the brainstem, confirming the main pathway of viral transport is through the vagus nerves. Sympathetic postganglionic neurons in the stellate and superior cervical ganglia were infected by 72 h, however, there was no evidence for retrograde transynaptic movement of the virus in sympathetic pathways in the central nervous system (CNS). These data demonstrate the organization of key structures within the CNS that receive afferent projections from the extrathoracic airways that likely play a role in the perception of airway sensations.

For many years the idea of a cough center in the brain dominated discussions in the field without any substantial progress in defining what this cough center is or how it functions. Substantial progress has now been made and many of the central neural elements involved in coughing are being described. Furthermore, hypothesis driven research into the function of these neural elements is providing exciting new leads for possible therapeutic targets. The concept of a specific, centrally acting drug for cough suppression is fast becoming a reality. This review summarizes the key findings from the past few years and provides a perspective on future directions for the development of novel antitussives.

In the present study, we describe a subset of nerve fibers, characterized by their immunoreactivity for the calcium-binding protein calretinin, that are densely and selectively associated with cholinergic postganglionic neurons in the guinea pig tracheal ganglia. Retrograde neuronal tracing with cholera toxin B, combined with immunohistochemical analyses, showed that these nerve fibers do not originate from sensory neurons in the nodose, jugular, or dorsal root ganglia or from motor neurons in the nucleus ambiguus, dorsal motor nucleus of the vagus nerve, spinal cord, stellate ganglia, or superior cervical ganglia. Calretinin-immunoreactive nerve fibers disappeared from tracheal segments after 48 h in organotypic culture, indicating that the fibers were of extrinsic origin. However, calretinin-positive nerve fibers persisted in tracheal ganglia when tracheae were cocultured with the adjacent esophagus intact. Immunohistochemical analysis of the esophagus revealed a population of cholinergic neurons in the esophageal myenteric plexus that coexpressed calretinin. In functional studies, electrical stimulation of the esophagus in vitro evoked measurable contractions of the trachea. These contractions were not altered by prior organotypic culture of the trachea and esophagus to remove the extrinsic innervation to the airways but were significantly ( P < 0.05) inhibited by the ganglionic blocker hexamethonium or by physical disruption of the tissue connecting the trachea and esophagus. These data suggest that a subset of esophageal neurons, characterized by the expression of calretinin and acetylcholine, provide a previously unrecognized excitatory input to tracheal cholinergic ganglia in guinea pigs.

The respiratory tree receives sensory innervation from the jugular and nodose vagal sensory ganglia. Neurons of these ganglia are derived from embryologically distinct origins and as such demonstrate differing molecular, neurochemical and physiological phenotypes. Furthermore, whereas nodose afferent neurons project to the nucleus of the solitary tract (nTS), recent neuroanatomical studies in rats suggest that jugular neurons have their central terminations in the paratrigeminal nucleus (Pa5). In the present study we confirm that guinea pigs demonstrate a comparable distinction between the brainstem terminations of nodose and jugular ganglia afferents. Thus, microinjection of fluorescently conjugated cholera toxin B (CT-B) neural tracers into the caudal nTS and Pa5 resulted in highly specific retrograde labelling of neurons in the nodose and jugular ganglia, respectively. Whereas nodose neurons more often expressed 160KD neurofilament proteins and the alpha3 subunit of Na+/K+ ATPase, significantly more jugular neurons expressed the neuropeptides substance P (SP) and, especially, Calcitonin Gene-Related Peptide (CGRP). Indeed, terminal fibers in the Pa5 compared to the nTS were characterized by their significantly greater expression of CGRP, further supporting the notion that jugular afferents project to trigeminal-related brainstem regions. Electrical stimulation of the guinea pig larynx following selective surgical denervation of the nodose afferent innervation to the larynx (leaving intact the jugular innervation) resulted in stimulus dependent respiratory slowing and eventual apnea. This jugular ganglia neuron mediated response was unaffected by bilateral microinjections of the GABAA agonist muscimol into the nTS, but was abolished by muscimol injected into the Pa5. Taken together these data confirm that jugular and nodose vagal ganglia afferent neurons innervate distinct central circuits and support the notion that multiple peripheral and central pathways mediate sensory responses associated with airway irritations.

Sensory nerves innervating the mucosa of the airways monitor the local environment for the presence of irritant stimuli and, when activated, provide input to the nucleus of the solitary tract (Sol) and paratrigeminal nucleus (Pa5) in the medulla to drive a variety of protective behaviors. Accompanying these behaviors are perceivable sensations that, particularly for stimuli in the proximal end of the airways, can be discrete and localizable. Airway sensations likely reflect the ascending airway sensory circuitry relayed via the Sol and Pa5, which terminates broadly throughout the CNS. However, the relative contribution of the Sol and Pa5 to these ascending pathways is not known. In the present study, we developed and characterized a novel conditional anterograde transneuronal viral tracing system based on the H129 strain of herpes simplex virus 1 and used this system in rats along with conventional neuroanatomical tracing with cholera toxin B to identify subcircuits in the brainstem and forebrain that are in receipt of relayed airway sensory inputs via the Sol and Pa5. We show that both the Pa5 and proximal airways disproportionately receive afferent terminals arising from the jugular (rather than nodose) vagal ganglia and the output of the Pa5 is predominately directed toward the ventrobasal thalamus. We propose the existence of a somatosensory-like pathway from the proximal airways involving jugular ganglia afferents, the Pa5, and the somatosensory thalamus and suggest that this pathway forms the anatomical framework for sensations arising from the proximal airway mucosa.

Irritations of the airways evoke brainstem-mediated reflex changes in breathing and complex sensorimotor responses dependent upon ascending inputs to higher brain centers. Airway vagal afferents te...

Described in this unit are methods for establishing guinea pig models of asthma. Sufficient detail is provided to enable investigators to study bronchoconstriction, cough, airway hyperresponsiveness, inflammation, and remodeling.

The airways and lungs are densely innervated by sensory nerves, which subserve multiple roles in both the normal physiological control of respiratory functions and in pulmonary defense. These sensory nerves are therefore not homogeneous in nature, but rather have physiological, molecular and anatomical phenotypes that reflect their purpose. All sensory neuron subtypes provide input to the central nervous system and drive reflex changes in respiratory and airway functions. But less appreciated is that ascending projections from these brainstem inputs to higher brain regions can also induce behavioural changes in respiration. In this brief review we provide an overview of the current understanding of airway sensory pathways, with specific reference to those involved in reflex and behavioural cough responses following airways irritation.

Subtypes of the hyperpolarization-activated cyclic nucleotide-gated (HCN) family of cation channels are widely expressed on nerves and smooth muscle cells in many organ systems, where they serve to regulate membrane excitability. Here we have assessed whether HCN channel inhibitors alter the function of airway smooth muscle or the neurons that regulate airway smooth muscle tone.The effects of the HCN channel inhibitors ZD7288, zatebradine and Cs(+) were assessed on agonist and nerve stimulation-evoked changes in guinea pig airway smooth muscle tone using tracheal strips in vitro, an innervated tracheal tube preparation ex vivo or in anaesthetized mechanically ventilated guinea pigs in vivo. HCN channel expression in airway nerves was assessed using immunohistochemistry, PCR and in situ hybridization.HCN channel inhibition did not alter airway smooth muscle reactivity in vitro to exogenously administered smooth muscle spasmogens, but significantly potentiated smooth muscle contraction evoked by the sensory nerve stimulant capsaicin and electrical field stimulation of parasympathetic cholinergic postganglionic neurons. Sensory nerve hyperresponsiveness was also evident in in vivo following HCN channel blockade. Cs(+) , but not ZD7288, potentiated preganglionic nerve-dependent airway contractions and over time induced autorhythmic preganglionic nerve activity, which was not mimicked by inhibitors of potassium channels. HCN channel expression was most evident in vagal sensory ganglia and airway nerve fibres.HCN channel inhibitors had a previously unrecognized effect on the neural regulation of airway smooth muscle tone, which may have implications for some patients receiving HCN channel inhibitors for therapeutic purposes.

Distinct parasympathetic postganglionic neurons mediate contractions and relaxations of the guinea pig airways. We set out to characterize the vagal inputs that regulate contractile and relaxant airway parasympathetic postganglionic neurons. Single and dual retrograde neuronal tracing from the airways and esophagus revealed that distinct, but intermingled, subsets of neurons in the compact formation of the nucleus ambiguus innervate these two tissues. Tracheal and esophageal neurons identified in the nucleus ambiguus were cholinergic. Esophageal projecting neurons also preferentially (greater than 70 percent) expressed the neuropeptide CGRP, but could not otherwise be distinguished immunohistochemically from tracheal projecting preganglionic neurons. Few tracheal or esophageal neurons were located in the dorsal motor nucleus of the vagus. Electrical stimulation of the vagi in vitro elicited stimulus dependent tracheal and esophageal contractions and tracheal relaxations. The voltage required to evoke tracheal smooth muscle relaxation was significantly higher than that required for evoking either tracheal contractions or esophageal longitudinal striated muscle contractions. Together our data support the hypothesis that distinct vagal preganglionic pathways regulate airway contractile and relaxant postganglionic neurons. The relaxant preganglionic neurons can also be differentiated from the vagal motor neurons that innervate the esophageal striated muscle.

Several indications suggest that supramedullary brain regions receive sensory information from the airways and provide motor control to the brainstem neurons that control coughing. However, the organization of this circuitry has not been described in any detail. In this short review we will discuss how state-of-the-art functional brain imaging techniques in humans and animals will enable unprecedented insights into the supramedullary brain regions that help control coughing. In addition we will describe the likely similarities between cough-related higher brain networks and those involved in the processing of other aversive sensory modalities, such as pain.

1. Cough is a primary defensive reflex that protects the airways from potentially harmful stimuli. 2. During many respiratory diseases, the cough reflex threshold is lowered and coughing becomes excessive. 3. Currently available therapeutics are mostly ineffective at suppressing excessive coughing. 4. In the present review, we describe the sensory neural pathways involved in cough, how these pathways may become dysfunctional in airway disease and the most recent advances that have been made in identifying future targets for cough suppression.

Coughing in humans is typically preceded by a desire (or urge) to cough. The neural circuitry involved in sensing airway irritation and generating the urge-to-cough in humans is essentially unknown.The aim of the present study was to use functional brain imaging to describe the supramedullary regions that are activated in humans during capsaicin inhalation.Experiments were performed on 10 healthy subjects (5 males, 5 females). Capsaicin doses were individually tailored to evoke a transient and reversible urge-to-cough. Blood oxygen level-dependent (BOLD) functional magnetic resonance measures were collected during repeated 24-second challenges with capsaicin or saline inhalation and subjects were asked to rate the urge-to-cough intensity of each challenge.Capsaicin inhalation reliably evoked an urge-to-cough, which was associated with activations in a variety of brain regions, including the insula cortex, anterior midcingulate cortex, primary sensory cortex, orbitofrontal cortex, supplementary motor area, and cerebellum.These data provide the first insights into the cortical neuronal network involved in sensing airway irritation and modulating coughing in humans.

Loop diuretics have been shown to inhibit cough and other airway defensive reflexes via poorly defined mechanisms. We test the hypothesis that the furosemide-sensitive Na+-K+-2Cl−cotransporter (NKCC1) is expressed by sensory nerve fibers innervating the airways where it plays an important role in regulating sensory neural activity. NKCC1 immunoreactivity was present on the cell membranes of most nodose and jugular ganglia neurons projecting to the trachea, and it was present on the peripheral terminals of putative mechanosensory nerve fibers in the airways. In urethane-anesthetized, spontaneously breathing guinea pigs, bolus application of citric acid (1 mM to 2 M) to an isolated and perfused segment of the tracheal mucosa evoked coughing and respiratory slowing. Removal of Cl−from the tracheal perfusate evoked spontaneous coughing and significantly potentiated cough and respiratory slowing reflexes evoked by citric acid. The NKCC1 inhibitor furosemide (10–100 μM) significantly reduced both the number of coughs evoked by citric acid and the degree of acid-evoked respiratory slowing ( P < 0.05). Localized tracheal pretreatment with the Cl−channel inhibitors DIDS or niflumic acid (100 μM) also significantly reduced cough, whereas the GABAAreceptor agonist muscimol potentiated acid-evoked responses. These data suggest that vagal sensory neurons may accumulate Cl−due to the expression of the furosemide-sensitive Cl−transporter, NKCC1. Efflux of intracellular Cl−, in part through calcium-activated Cl−channels, may play an important role in regulating airway afferent neuron activity.

There is an overwhelming body of evidence to support the existence of higher brain circuitries involved in the sensory detection of airways irritation and the motor control of coughing. The concept that cough is purely a reflex response to airways irritation is now superseded by the recognition that perception of an urge-to-cough and altered behavioral modification of coughing are key elements of cough disorders associated with airways disease. Understanding the pathways by which airway sensory nerves ascend into the brain and the patterns of neural activation associated with airways irritation will undoubtedly provide new insights into disordered coughing. This brief review aims to explore our current understanding of higher order cough networks by summarizing data from recent neuroanatomical and functional studies in animals and humans. We provide evidence for the existence of distinct higher order network components involved in the discrimination of signals arising from the airways and the motor control of coughing. The identification of these network components provides a blueprint for future research and the development of targeted managements for cough and the urge-to-cough.

Background Cough in guinea pigs is mediated in part by capsaicin-insensitive low threshold mechanoreceptors (cough receptors). Functional studies suggest that cough receptors represent a homogeneous population of nodose ganglia-derived sensory neurons. In the present study we set out to characterize the neurochemical profile of cough receptor neurons in the nodose ganglia. Methods Nodose neurons projecting to the guinea pig trachea were retrogradely labeled with fluorogold and processed immunohistochemically for the expression of a variety of transporters (Na+/K+/2C1- co-transporter (NKCC1), α1 and α3 Na+/K+ ATPase, vesicular glutamate transporters (vGlut)1 and vGlut2), neurotransmitters (substance P, calcitonin gene-related peptide (CGRP), somatostatin, neuronal nitric oxide synthase (nNOS)) and cytosolic proteins (neurofilament, calretinin, calbindin, parvalbumin). Results Fluorogold labeled ~3 per cent of neurons in the nodose ganglia with an average somal perimeter of 137 ± 6.2 μm (range 90–200 μm). All traced neurons (and seemingly all nodose neurons) were immunoreactive for NKCC1. Many (> 90 per cent) were also immunoreactive for vGlut2 and neurofilament and between 50 and 85 per cent expressed α1 ATPase, α3 ATPase or vGlut1. Cough receptor neurons that did not express the above markers could not be differentiated based on somal size, with the exception of neurofilament negative neurons which were significantly smaller (P < 0.05). Less than 10 per cent of fluorogold labeled neurons expressed substance P or CGRP (and these had somal perimeters less than 110 μm) and none expressed somatostatin, calretinin, calbindin or parvalbumin. Two distinct patterns of nNOS labeling was observed in the general population of nodose neurons: most neurons contained cytosolic clusters of moderately intense immunoreactivity whereas less than 10 per cent of neurons displayed uniform intensely fluorescent somal labeling. Less than 3 per cent of the retrogradely traced neurons were intensely fluorescent for nNOS (most showed clusters of nNOS immunoreactivity) and nNOS immunoreactivity was not expressed by cough receptor nerve terminals in the tracheal wall. Conclusion These data provide further insights into the neurochemistry of nodose cough receptors and suggest that despite their high degree of functional homogeneity, nodose cough receptors subtypes may eventually be distinguished based on neurochemical profile.