1 SCIENTIFIC REPORTS | (2019) 9:16982 | https://doi.org/10.1038/s41598-019-53684-3 www.nature.com/scientificreports The autonomic innervation of hairy skin in humans: an in vivo confocal study Vincenzo Donadio 1* , Alex Incensi 1 , Veria Vacchiano 1,2 , Rossella Infante 1,2 , Martina Magnani 1 & Rocco Liguori 1,2 The autonomic innervation of the skin includes diferent subsets of adrenergic and cholinergic fbers both in humans and animals. The corresponding chemical code is complex and often difcult to ascertain. Accordingly, a detailed histochemical description of skin autonomic fber subtypes is lacking in humans. To characterize skin autonomic nerve subtypes may help to better understand the selective damage of specifc skin autonomic fbers afecting human diseases such as the adrenergic fbers directed to skin vessels in Parkinson’s disease or the cholinergic sudomotor fbers in Ross Syndrome. The present study aimed at characterizing subtypes of autonomic fbers in relation to their target organs by means of an immunofuorescent technique and confocal microscopy. We studied 8 healthy subjects (5 males and 3 females) aged 45 ± 2 (mean ± SE) years without predisposing causes for peripheral neuropathy or autonomic disorders. They underwent skin biopsy from proximal (thigh) and distal (leg) hairy skin. A combination of adrenergic (i.e. tyrosine-hydroxylase- TH and dopamine beta-hydroxylase- DbH) and cholinergic (vesicular acetylcholine transporter- VACHT) autonomic markers and neuropeptidergic (i.e. neuropeptide Y- NPY, calcitonin gene-related peptide- CGRP, substance P- SP, and vasoactive intestinal peptide- VIP) markers were used to characterize skin autonomic fbers. The analysed skin autonomic structures included: 58 sweat glands, 91 skin arterioles and 47 arrector pili muscles. Our results showed that all skin structures presented a sympathetic adrenergic but also cholinergic innervation although in diferent proportions. Sympathetic adrenergic fbers were particularly abundant around arterioles and arrector pili muscles whereas sympathetic cholinergic fbers were mainly found around sweat glands. Neuropeptides were diferently expressed in sympathetic fbers: NPY were found in sympathetic adrenergic fbers around skin arterioles and very seldom sweat glands but not in adrenergic fbers of arrector pili muscles. By contrast CGRP, SP and VIP were expressed in sympathetic cholinergic fbers. Cholinergic fbers expressing CGRP, SP or VIP without TH or DbH staining were found in arterioles and arrector pili muscles and they likely represent parasympathetic fbers. In addition, all skin structures contained a small subset of neuropeptidergic fbers devoid of adrenergic and cholinergic markers with a likely sensory function. No major diferences were found between males and females and proximal and distal sites. In summary hairy skin contains sympathetic adrenergic and cholinergic fbers diferently distributed around skin structures with a specifc distribution of neuropeptides. The autonomic skin innervation also contains a small amount of fbers, likely to be parasympathetic and sensory. Te autonomic innervation of hairy skin includes diferent subsets of adrenergic and cholinergic fbers both in humans and animals but the chemical code of autonomic nerve terminals is complex and not well known 1–4 . Tis is unfortunate since the neuropeptide content of a fber subpopulation is likely to defne important aspects of its functional characteristics. Other such aspects are related to the fring characteristics of the eferent nerve fbres (which can be studied by microneurography 5,6 ) but the link between the neurochemical codes of eferent C fber populations and their fring patterns is unknown making the interpretation of frequently used autonomic tests difcult 7,8 . A better knowledge of these factors and relationships may also lead to a better understanding of the selective damage of autonomic fbers in human diseases, such as Parkinson´s disease 9–11 or Ross syndrome 12,13 . 1 IRCCS Istituto delle Scienze Neurologiche di Bologna, UOC Clinica Neurologica, Bologna, Italy. 2 Dipartimento di Scienze Biomediche e Neuromotorie, Università di Bologna, Bologna, Italy. *email: vincenzo.donadio@unibo.it OPEN