Selective visualisation of sensory receptors in the smooth muscle layer of ex-vivo airway whole-mounts by styryl pyridinium dyes

Recently, we established the location, morphology and neurochemical coding of vagal smooth-muscle-associated airway receptors (SMARs) in rat lungs. These receptors were characterised as branching laminar terminals that originated from myelinated nerve fibres and were intercalated between airway smooth-muscle bundles. To allow the direct physiological examination of these receptors, the present investigation aimed at visualising SMARs in airway whole-mounts of rat and mouse lungs ex vivo. Short incubation with various styryl pyridinium dyes (AM1-43, FM2-10, FM4-64 or 4-Di-2-ASP) gave a highly selective fluorescent visualisation of both laminar nerve terminals and myelinated fibres from which they originated throughout the intrapulmonary airway tree in mouse and in rat. The reliable and specific labelling of SMARs ex vivo with these lipophilic membrane dyes was confirmed via immunostaining for protein gene-product 9.5 and vesicular glutamate transporters. Similar to the intrapulmonary location of NEBs, these SMARs appeared to be even more explicitly located near airway bifurcations. Both the trachealis muscle and the smooth-muscle bundles of extrapulmonary bronchi were also shown to contain laminar nerve terminals that were morphologically similar to the SMARs reported in the intrapulmonary airways. Thus, this study provides an in-vitro model enabling, for the first time, the fast and reliable visualisation of SMARs and the myelinated nerve fibres from which they originate in airway whole-mount preparations ex vivo. As such, this model opens up further perspectives and creates a valid basis for direct physiological measurement and manipulation of the individually identified airway receptors.