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

Stuart B Mazzone,Alice E Mcgovern

doi:10.1186/1745-9974-4-9

Abstract

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.

Highlights

Cough in guinea pigs is mediated in part by capsaicin-insensitive low threshold mechanoreceptors
Extensive electrophysiological analyses of the activation profiles of nodose neurons projecting to the guinea pig trachea and larynx suggests that the majority of these neurons form a seemingly homogeneous population of neurons that display the functional characteristics of cough receptors [1,2]
The wound was sutured and the animals were allowed to recover for 7 days at which time they were anesthetized with sodium pentobarbital (100 mg/kg i.p.) and transcardially perfused with 10 mM phosphate buffered saline (PBS) followed by 4% paraformaldehyde in PBS

Summary

Introduction

Cough in guinea pigs is mediated in part by capsaicin-insensitive low threshold mechanoreceptors (cough receptors). Previous studies have identified a novel vagal sensory nerve subtype that innervates the large airways (larynx, trachea and main bronchi) of guinea pigs and is likely responsible for defensive cough in this species [1] These sensory neurons (referred to as cough receptors) are derived from the nodose ganglia and are characterized by their insensitivity to capsaicin and their sensitivity to both rapid reductions in pH and punctuate (touch-like) mechanical stimulation [1,2,3]. Unlike other classically defined low threshold mechanoreceptors which innervate the airways and lungs, cough receptors display a low sensitivity to mechanical stretch (including inflation/ deflation and bronchospasm), conduct action potentials slower (~5 m/sec for cough receptors compared to > 15 m/sec for intrapulmonary stretch receptors) and are unresponsive to the purinergic agonist α,β-methylene ATP [1] Based on these observations, cough receptors are believed to represent a distinct airway afferent nerve in this species (reviewed in [4]). In the guinea pig trachea and larynx, there are very few nodose capsaicin-sensitive nociceptors (tracheal nociceptors are mostly derived from the jugular vagal ganglia) and no classically defined rapidly adapting or slowly adapting stretch receptors [1,2]

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