Alice E. McGovern

Transneuronal tracing of airways-related sensory circuitry using herpes simplex virus 1, strain H129

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.

Immunohistochemical characterization of nodose cough receptor neurons projecting to the trachea of guinea pigs

BackgroundCough 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.MethodsNodose 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).ResultsFluorogold 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.ConclusionThese 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.

Mapping supramedullary pathways involved in cough using functional brain imaging: Comparison with pain

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.

Sensorimotor circuitry involved in the higher brain control of coughing.

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.

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.

Anterograde neuronal circuit tracing using a genetically modified herpes simplex virus expressing EGFP.

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.

Evidence for multiple sensory circuits in the brain arising from the respiratory system: an anterograde viral tract tracing study in rodents

Complex sensations accompany the activation of sensory neurons within the respiratory system, yet little is known about the organization of sensory pathways in the brain that mediate these sensations. In the present study, we employ anterograde viral neuroanatomical tract tracing with isogenic self-reporting recombinants of HSV-1 strain H129 to map the higher brain regions in receipt of vagal sensory neurons arising from the trachea versus the lungs, and single-cell PCR to characterize the phenotype of sensory neurons arising from these two divisions of the respiratory tree. The results suggest that the upper and lower airways are predominantly innervated by sensory neurons derived from the somatic jugular and visceral nodose cranial ganglia, respectively. This coincides with central circuitry that is predominately somatic-like, arising from the trachea, and visceral-like, arising from the lungs. Although some convergence of sensory pathways was noted in preautonomic cell groups, this was notably absent in thalamic and cortical regions. These data support the notion that distinct afferent subtypes, via distinct central circuits, subserve sensations arising from the upper versus lower airways. The findings may explain why sensations arising from different levels of the respiratory tree are qualitatively and quantitatively unique.

Characterization of the Vagal Motor Neurons Projecting to the Guinea Pig Airways and Esophagus

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 (nAmb) innervate these two tissues. Tracheal and esophageal neurons identified in the nAmb were cholinergic. Esophageal projecting neurons also preferentially (greater than 70%) 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.

The Role of the Paratrigeminal Nucleus in Vagal Afferent Evoked Respiratory Reflexes: A Neuroanatomical and Functional Study 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 labeling of neurons in the nodose and jugular ganglia, respectively. Whereas, nodose neurons more often expressed 160 KD 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.

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.