Proceedings of The Physiological Society
University of Cambridge (2008) Proc Physiol Soc 11, C73
Characterisation of epidermal primary afferents in the mouse.
G. Bruce1, R. Barrett-Jolley1, R. Morris1
1. Veterinary Preclinical Sciences, University of Liverpool, Liverpool, United Kingdom.
The epidermis is richly innervated with sensory afferents most of which are of the “non-peptidergic” group (Zylka et al., 2005; Belle et al., 2007) and in the present study some of the characteristics of these afferents have been determined. Studies were conducted on C57/Bl6 mice (SA36 strain) carrying the thy1.2-egfp gene expressed in non-peptide epidermal afferents (Belle et al., 2007). For anatomy, animals were terminally anaesthetised with sodium pentobarbitone i.p. (80mg/kg) prior to whole body perfusion fixation with 4% paraformaldehyde and for electrophysiology they were anaesthetised with halothane and killed by decapitation. Dorsal root ganglion (DRG) neurones were either studied shortly following acute dissociation (AD) within 2-9 hours of plating or after maintaining in short-term culture (SC) between 1-3 days post-plating. The responses of eGFP positive neurones recorded in whole-cell current-clamp mode to depolarising and hyperpolarising current pulses were investigated. All results given are mean ± S.E.M and statistical analysis between cell types was done by one-way ANOVA with Tukey post test unless otherwise stated. Immunoreactivity for P2X3 (mean surface area (m.s.a.) 444.3 ± 4.43µm2, n=800) was observed in 85.1% of eGFP-positive neurones (m.s.a. 509.9 ± 7.32µm2, n=737), whilst TRPV1 immunoreactivity (m.s.a. 358.1 ± 15.7µm2, n=179) marked a separate, smaller population of neurones only 5.02% of which expressed eGFP (1.62% of eGFP population). The mean size of the AD eGFP-positive neurones (n=32) (mean soma diameter 26.1 ± 0.76µm) was larger than those in SC (n=52) (mean soma diameter 21.31 ± 0.42µm, p < 0.001). This could be due to shrinkage following loss of axonal arborisation or be due to selective loss of small neurones that may not have settled in the acutely dissociated preparations. Their firing patterns to depolarising pulses could also be separated into five distinct groups burst (AD 12.5%, SC 3.8%), delayed (AD 3.1%, SC 0%), phasic (AD 9.3%, SC 1.9%), transient (AD 68.7%, SC 50%) and tonic firing (AD 6.2%, SC 44.2%). Action potential overshoot magnitudes were similar under the two conditions (AD 28.14 ± 2.12mV, SC 28.96 ± 1.39mV, p > 0.05) but action potentials were broader in SC neurones (AD 6.59 ± 1.1ms, SC 9.88 ± 0.85ms, p < 0.05) and more frequently had inflections on their repolarising slope. Approximately half of the AD neurones (53.13%) displayed delayed rectification and a rebound spike when hyperpolarised suggesting the HCN mediated current, Ih, is present. These results are consistent with the literature, showing that non-peptidergic primary afferents innervating the epidermis in the mouse are sensitive to ATP but not to capsaicin. Whilst some display characteristics of nociceptors (broad, inflected action potentials) others share properties that have been reported in cold sensitive neurones (Ih).
Where applicable, experiments conform with Society ethical requirements