P1.01. DEEP BODY PEPTIDERGIC AFFERENTS LACK ADVILLIN EXPRESSION: IMPLICATIONS FOR SENSORY NEURON PROFILING
Julia Niemczycka1, Joanna Bernacka1, Douglas M. Lopes2, Kirsty Bannister3, Mateusz Kucharczyk1,4
1 Łukasiewicz Research Network - PORT Polish Center for Technology Development, ul. Stabłowicka 147, 54-066, Wrocław, Poland
2 University College London, Department of Neuromuscular Diseases, Gower Street, London WC1E 6BT, UK
3 Imperial College London, Department of Life Sciences, South Kensington, London SW7 2AZ, UK
4 King’s College London, Wolfson Sensory, Pain and Regeneration Centre, London SE1 1UL, UK
INTRODUCTION: Heterogenous population of peripheral neurons is housed in dorsal root ganglia (DRG). Those diverse somatosensory afferent neurons mediate key bodily sensations ranging from touch and proprioception to temperature and pain. Advillin (Avil), an actin-binding protein, is a commonly used marker of all sensory and sympathetic neurons. However, recent findings question its universality, particularly in certain peptidergic subpopulations.
AIM(S): To evaluate which afferents are not covered by Avil and thus could exhibit new interesting population of sensory neurons.
METHOD(S): We used three transgenic mouse lines: Avil-eGFP labelling majority of sensory and sympathetic neurons, Calca-eGFP labelling peptidergic nociceptors, and ChRNA3-eGFP labelling sympathetic neurons and ‘silent nociceptors’, a unique subpopulation of peptidergic nociceptors. Sensory neurons innervating discreet bodily compartments (i.e. tibia, knee, bladder, hind paw) were retrogradely labelled with Fast Blue. Lumbar and sacral DRG were cryosectioned and immunostained for sensory markers (i.e., CGRP, Avil, Tubulin-βIII, IB4). Selected adrenoceptors were analysed in situ with RNAscope assay also. Following confocal microscopy, detailed colocalization analysis was performed. Additionally, representative DRG were optically cleared using PACT protocol and imaged with confocal and light-sheet microscopes. Image analysis was done in Fiji and Imaris, while quantification data was analysed by custom Python scripts and GraphPad Prism.
RESULTS: We demonstrate that Avil does not universally cover sensory neurons. Specifically, a subset of peptidergic afferents enriched in CGRP and innervating deep tissues exhibits low or undetectable levels of Avil. Data is supported by post hoc snRNAseq analysis.
CONCLUSIONS: Avil is not a universal marker of sensory neurons, biased towards superficial skin afferents.
FINANCIAL SUPPORT: Funded by National Science Centre grant (2022/D/NZ4/02676), held by M. Kucharczyk.