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Abstracts / Autonomic Neuroscience: Basic and Clinical 149 (2009) 1126

Acknowledgement: we thank Dr Catherine Bennett for her

assistance with the design of the questionnaire.

P9.2 Sensory pathways from the genital tract to lumbar and sacral
autonomic outflows in female guinea-pigs

doi:10.1016/j.autneu.2009.05.238

I.L. Gibbins, S.Y. Yuan, P.I Vilimas, V.P. Zagorodnyuk, J.L. Morris (Centre
for Neuroscience, Flinders University, GPO Box 2100, Adelaide, SA
5001, Australia)

P9 Sensory
P9.1 Mechanogated two-pore-domain potassium channels in
murine lungs: Special focus on sensory airway receptors
R. Lembrechts, I. Brouns, I. Pintelon, K. Schnorbusch, J.-P. Timmermans,
D. Adriaensen (Laboratory of Cell Biology and Histology, Department
of Veterinary Sciences, University of Antwerp, Groenenborgerlaan 171,
BE-2020, Antwerp, Belgium)
Over the past few years, we reported data suggesting the potential
involvement of pulmonary neuroepithelial bodies (NEBs) and smooth
muscle associated airway receptors (SMARs) in the transduction of
mechanical stimuli from the airways. NEBs are structurally well-defined
receptoreffector end organs composed of densely innervated groups of
neuroendocrine cells, located in the epithelium of intrapulmonary
airways. Due to their complex innervation pattern, a role as sensor for
various stimuli in the airways is strongly suggested. NEBs receive at least
two different populations of myelinated vagal afferent nerve terminals
that can be discriminated based on the expression of P2X2/3 and
VGLUT1 immunoreactivity, respectively. Both populations reveal a
neurochemical coding indicative of a mechanosensory function. SMARs
originate from myelinated vagal sensory nerve fibers and give rise to
laminar nerve endings in the smooth muscle bundles at different levels
of the airways. SMARs have a neurochemical coding that is, similar to
that of nerve fibers contacting NEBs, suggestive of a mechanosensitive
function. Despite this extensive morphological characterization, physiological evidence for a role of NEBs and SMARs as airway receptors is
still lacking. Electrophysiological fiber recordings in the vagus nerve
revealed that the majority of myelinated pulmonary afferents is
mechanosensitive, suggesting that both NEBs and SMARs may represent
the morphological counterparts of at least subpopulations of the
electrophysiologically identified airway mechanoreceptors. In the
present study, we focused on the expression of mechanogated twopore-domain K+ (K2P) channels in mouse airways, with special
reference to the NEB micro-environment and SMARs. TREK and TRAAK
channels, which belong to the large family of K2P-channels, are known
to be activated by mechanical stimuli such as elevated pressure on cell
membranes, cell stretch and cell swelling. We investigated the
expression of these mechanogated K2P-channels by multiple immunostaining. TREK-1 immunoreactivity was found in the lungs but
seemed to be restricted to smooth muscle cells surrounding the airways
and blood vessels of the lung. TRAAK appeared to be mainly expressed in
the extensive intraepithelial terminals of both the P2X2/3- and VGLUT/
CB-immunoreactive vagal sensory nerve fibers that selectively contact
NEBs, and in SMARs that are located in the airway smooth muscle layer,
often close to the epithelium and NEBs. The observation that the
extensive terminals of vagal myelinated afferents in NEBs express
mechanogated K2P channels and hence harbor intrinsic mechanosensitive properties strengthens our hypothesis that, in addition to SMARs,
the NEB micro-environment likely harbours subpopulations of the
electrophysiologically characterized vagal airway mechanosensors.
Moreover, the present data suggest that NEBs may indeed be involved
in the transduction of mechanical changes in the airways.
Support: FWO grants G.0085.04 and G.0081.08 (D.A.); UA grants
GOA BOF 2007 (D.A.) and KP BOF 2006 (I.B.).

doi:10.1016/j.autneu.2009.05.239

The vasculature of the female genital tract is innervated by

vasodilator neurons with cell bodies located mainly in the paracervical ganglia. These neurons receive convergent preganglionic
inputs from sacral and lumbar segments of the spinal cord.
Mechanical stimulation of the lower urinogenital tract activates a
spinal reflex to the paracervical ganglia but the organisation of these
pathways is largely unknown. Here we have used a confocal 3D
microscopy to identify sensory endings in the genital skin, and a
novel in vitro preparation of isolated perfused spinal cord and
peripheral nerves to investigate functional pathways between
sensory inputs from and autonomic outputs to the pelvic viscera.
For confocal microscopy, young adult female guinea-pigs were
killed by stunning or a lethal overdose of urethane anaesthetic. The
whole perinealpudendal region including the lower urinogenital
and digestive tracts, was removed, hemisected in the sagittal plane,
fixed in 0.5% picric acid / 4% formaldehyde, and prepared for
immunohistochemistry. Sagittal cryostat sections were cut through
the whole blocks which were triple-labelled for immunoreactivity for
neuron-specific enolase (NSE, a general neuronal marker), calcitonin
gene-related peptide (CGRP), and substance P (SP). 3D image sets
were gathered on a Leica SP5 confocal microscope and reconstructed
with Avizo software. For functional studies, the isolated spinal cord
and attached peripheral nerves were perfused in vitro with HEPESbuffered balanced salt solution containing 1.25% Ficoll. Descending
spinal pathways were stimulated electrically with a pair of electrodes
inserted into the spinal cord. The pudendal nerve was stimulated
with a suction electrode. Compound action potentials were recorded
via suction electrodes on pelvic, splanchnic or hypogastric nerves
with PowerLab Chart.
Confocal microscopy revealed extraordinarily dense, complex
sensory innervation of the genital and surrounding perineal skin.
Dermal papillae in non-hairy skin of the clitoris and vulva were
packed with large diameter specialised endings, presumably representing low-threshold mechanoceptors. Each cluster of endings
contains at least one fine varicose fibre containing CGRP but not SP.
Other areas of the perineal skin lacked these large endings, but were
characterised by tufts of fine NSE fibres mostly lacking peptides.
Within the vagina-cervix, most subepithelial sensory fibres contained
both CGRP and SP, and intra-epithelial fibres of any type were mostly
absent.
Stimulation of the lower thoracic spinal cord caused compound
action potentials in the hypogastric nerves confirming the viability of
the preparation. These responses were abolished by transection of
the lumbar splanchnic nerves. Simulation of the pudendal nerve
resulted in compound action potentials recorded in the pelvic nerves.
Pudendal nerve stimulation also generated compound action potentials in the lumbar splanchnic and hypogastric nerves, indicating the
existence of an ascending spinal pathway from sacral to lumbar
spinal segments. Transmission along this pathway was enhanced by
bicuculline (10 M).
These results reveal a sacral-to-lumbar spinal circuit that can
activate autonomic outflow to the pelvic viscera, presumably mostly
involving vasodilator pathways. Inputs to this circuit include large
myelinated and fine unmyelinated sensory neurons that probably
have overlapping receptive fields. Their close association may
contribute to genital pain syndromes.
doi:10.1016/j.autneu.2009.05.249