Evidence for vagal sensory neural involvement in influenza pathogenesis and disease.

Influenza A virus (IAV) is a common respiratory pathogen and a global cause of significant and often severe morbidity. Although inflammatory immune responses to IAV infections are well described, little is known about how neuroimmune processes contribute to IAV pathogenesis. In the present study, we employed surgical, genetic, and pharmacological approaches to manipulate pulmonary vagal sensory neuron innervation and activity in the lungs to explore potential crosstalk between pulmonary sensory neurons and immune processes. Intranasal inoculation of mice with H1N1 strains of IAV resulted in stereotypical antiviral lung inflammation and tissue pathology, changes in breathing, loss of body weight and other clinical signs of severe IAV disease. Unilateral cervical vagotomy and genetic ablation of pulmonary vagal sensory neurons had a moderate effect on the pulmonary inflammation induced by IAV infection, but significantly worsened clinical disease presentation. Inhibition of pulmonary vagal sensory neuron activity via inhalation of the charged sodium channel blocker, QX-314, resulted in a moderate decrease in lung pathology, but again this was accompanied by a paradoxical worsening of clinical signs. Notably, vagal sensory ganglia neuroinflammation was induced by IAV infection and this was significantly potentiated by QX-314 administration. This vagal ganglia hyperinflammation was characterized by alterations in IAV-induced host defense gene expression, increased neuropeptide gene and protein expression, and an increase in the number of inflammatory cells present within the ganglia. These data suggest that pulmonary vagal sensory neurons play a role in the regulation of the inflammatory process during IAV infection and suggest that vagal neuroinflammation may be an important contributor to IAV pathogenesis and clinical presentation. Targeting these pathways could offer therapeutic opportunities to treat IAV-induced morbidity and mortality.

The impact of influenza pulmonary infection and inflammation on vagal bronchopulmonary sensory neurons.

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.

Descending Modulation of Laryngeal Vagal Sensory Processing in the Brainstem Orchestrated by the Submedius Thalamic Nucleus.

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 STATEMENT Jugular 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.

A role for neurokinin 1 receptor expressing neurons in the paratrigeminal nucleus in bradykinin-evoked cough in guinea-pigs.

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.

Translational review: Neuroimmune mechanisms in cough and emerging therapeutic targets.

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.

Central mechanisms of airway sensation and cough hypersensitivity.

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.

Distinct brainstem and forebrain circuits receiving tracheal sensory neuron inputs revealed using a novel conditional anterograde transsynaptic viral tracing system.

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.

Comparative localization of colorectal sensory afferent central projections in the mouse spinal cord dorsal horn and caudal medulla dorsal vagal complex.

The distal colon and rectum (colorectum) are innervated by spinal and vagal afferent pathways. The central circuits into which vagal and spinal afferents relay colorectal nociceptive information remain to be comparatively assessed. To address this, regional colorectal retrograde tracing and colorectal distension (CRD)-evoked neuronal activation were used to compare the circuits within the dorsal vagal complex (DVC) and dorsal horn (thoracolumbar [TL] and lumbosacral [LS] spinal levels) into which vagal and spinal colorectal afferents project. Vagal afferent projections were observed in the nucleus tractus solitarius (NTS), area postrema (AP), and dorsal motor nucleus of the vagus (DMV), labeled from the rostral colorectum. In the NTS, projections were opposed to catecholamine and pontine parabrachial nuclei (PbN)-projecting neurons. Spinal afferent projections were labeled from rostral through to caudal aspects of the colorectum. In the dorsal horn, the number of neurons activated by CRD was linked to pressure intensity, unlike in the DVC. In the NTS, 13% ± 0.6% of CRD-activated neurons projected to the PbN. In the dorsal horn, at the TL spinal level, afferent input was associated with PbN-projecting neurons in lamina I (LI), with 63% ± 3.15% of CRD-activated neurons in LI projecting to the PbN. On the other hand, at the LS spinal level, only 18% ± 0.6% of CRD-activated neurons in LI projected to the PbN. The collective data identify differences in the central neuroanatomy that support the disparate roles of vagal and spinal afferent signaling in the facilitation and modulation of colorectal nociceptive responses.

Investigation of vagal sensory neurons in mice using optical vagal stimulation and tracheal neuroanatomy.

In rats and guinea pigs, sensory innervation of the airways is derived largely from the vagus nerve, with the extrapulmonary airways innervated by Wnt1+ jugular neurons and the intrapulmonary airways and lungs by Phox2b+ nodose neurons; however, our knowledge of airway innervation in mice is limited. We used genetically targeted expression of enhanced yellow fluorescent protein-channelrhodopsin-2 (EYFP-ChR2) in Wnt1+ or Phox2b+ tissues to characterize jugular and nodose-mediated physiological responses and airway innervation in mice. With optical stimulation, Phox2b+ vagal fibers modulated cardiorespiratory function in a frequency-dependent manner while right Wnt1+ vagal fibers induced a small increase in respiratory rate. Mouse tracheae contained sparse Phox2b-EYFP fibers but dense networks of Wnt1-EYFP fibers. Retrograde tracing from the airways showed limited tracheal innervation by the jugular sensory neurons, distinct from other species. These differences in physiology and vagal sensory distribution have important implications when using mice for studying airway neurobiology.

Brainstem processing of cough sensory inputs in chronic cough hypersensitivity.

BACKGROUND: Chronic cough is a prevalent and difficult to treat condition often accompanied by cough hypersensitivity, characterised by cough triggered from exposure to low level sensory stimuli. The mechanisms underlying cough hypersensitivity may involve alterations in airway sensory nerve responsivity to tussive stimuli which would be accompanied by alterations in stimulus-induced brainstem activation, measurable with functional magnetic resonance imaging (fMRI). METHODS: We investigated brainstem responses during inhalation of capsaicin and adenosine triphosphate (ATP) in 29 participants with chronic cough and 29 age- and sex-matched controls. Psychophysical testing was performed to evaluate individual sensitivities to inhaled stimuli and fMRI was used to compare neural activation in participants with cough and control participants while inhaling stimulus concentrations that evoked equivalent levels of urge-to-cough sensation. FINDINGS: Participants with chronic cough were significantly more sensitive to inhaled capsaicin and ATP and showed a change in relationship between urge-to-cough perception and cough induction. When urge-to-cough levels were matched, participants with chronic cough displayed significantly less neural activation in medullary regions known to integrate airway sensory inputs. By contrast, neural activations did not differ significantly between the two groups in cortical brain regions known to encode cough sensations whereas activation in a midbrain region of participants with chronic cough was significantly increased compared to controls. INTERPRETATION: Cough hypersensitivity in some patients may occur in brain circuits above the level of the medulla, perhaps involving midbrain regions that amplify ascending sensory signals or change the efficacy of central inhibitory control systems that ordinarily serve to filter sensory inputs. FUNDING: Supported in part by a research grant from Investigator-Initiated Studies Program of Merck Sharp & Dohme Pty Ltd. The opinions expressed in this paper are those of the authors and do not necessarily represent those of Merck Sharp & Dohme (Australia) Pty Ltd.

Loss of NEDD4 causes complete XY gonadal sex reversal in mice.

Gonadogenesis is the process wherein two morphologically distinct organs, the testis and the ovary, arise from a common precursor. In mammals, maleness is driven by the expression of Sry. SRY subsequently upregulates the related family member Sox9 which is responsible for initiating testis differentiation while repressing factors critical to ovarian development such as FOXL2 and ß-catenin. Here, we report a hitherto uncharacterised role for the ubiquitin-protein ligase NEDD4 in this process. XY Nedd4-deficient mice exhibit complete male-to-female gonadal sex reversal shown by the ectopic upregulation of Foxl2 expression at the time of gonadal sex determination as well as insufficient upregulation of Sox9. This sex reversal extends to germ cells with ectopic expression of SYCP3 in XY Nedd4-/- germ cells and significantly higher Sycp3 transcripts in XY and XX Nedd4-deficient mice when compared to both XY and XX controls. Further, Nedd4-/- mice exhibit reduced gonadal precursor cell formation and gonadal size as a result of reduced proliferation within the developing gonad as well as reduced Nr5a1 expression. Together, these results establish an essential role for NEDD4 in XY gonadal sex determination and development and suggest a potential role for NEDD4 in orchestrating these cell fate decisions through the suppression of the female pathway to ensure proper testis differentiation.

Jugular vagal ganglia neurons and airway nociception: A target for treating chronic cough.

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.

Mini Review: Central Organization of Airway Afferent Nerve Circuits.

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.

Modulation of Vagal Sensory Neurons via High Mobility Group Box-1 and Receptor for Advanced Glycation End Products: Implications for Respiratory Viral Infections.

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 primarily via sensory 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.

Innervation of tracheal parasympathetic ganglia by esophageal cholinergic neurons: evidence from anatomic and functional studies in guinea pigs.

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.

Peripheral and central mechanisms of cough hypersensitivity.

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.

Local D2- to D1-neuron transmodulation updates goal-directed learning in the striatum.

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 that mediate goal-directed control are reorganized during new learning remains unknown. Here, by mapping a dopamine-dependent transcriptional activation marker in large ensembles of spiny 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 transmodulation across the 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.

Transcriptional Profiling of Individual Airway Projecting Vagal Sensory Neurons.

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.

Hippocampal modulation of cardiorespiratory function.

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.

Reflex regulation of breathing by the paratrigeminal nucleus via multiple bulbar circuits.

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.