Contents lists available at ScienceDirect Archives of Oral Biology journal homepage: www.elsevier.com/locate/archoralbio Tyrosine-hydroxylase, dopamine β-hydroxylase and choline acetyltransferase-like immunoreactive fibres in the human major sublingual gland Francesco Loy a , Maria Pina Serra a , Marianna Boi a , Raffaella Isola a , Jörgen Ekström b , Marina Quartu a, ⁎ a Section of Cytomorphology, Department of Biomedical Sciences, University of Cagliari, Cittadella Universitaria di Monserrato, 09042 Monserrato CA, Italy b Department of Pharmacology, Institute of Neuroscience and Physiology, The Sahlgrenska Academy at University of Gothenburg, Medicinaregatan 13, Box 431, 405 30 Göteborg, Sweden ARTICLE INFO Keywords: Human major sublingual gland Tyrosine hydroxylase Dopamine beta-hydroxylase Choline acetyltransferase Immunohistochemistry ABSTRACT Objective: To study the innervation of the major sublingual gland by means of immunohistochemistry. Design: Bioptic and autoptic specimens of the major sublingual gland of humans were examined for the presence of immunoreactivity to tyrosine hydroxylase and dopamine-β-hydroxylase, on one hand, and choline acetyl- transferase, on the other, to indicate adrenergic and cholinergic nerves, respectively. Results: Acini and ducts were supplied by both divisions of the autonomic nervous system. Conclusions: Mucous and seromucous cells of the human major sublingual glands may respond with secretion not only to parasympathetic activity but also to sympathetic activity. The major sublingual gland is therefore a potential contributor to the mucin secretion recently reported in the literature in response to high sympathetic activity during physical exercise. 1. Introduction In humans, the sublingual gland tissue mass is not a single organ but consists of a large segment, the major sublingual gland, and a cluster of up to 30 small independent glands, the minor sublingual glands (Riva, Tandler, & Testa Riva, 1988; Riva, Loffredo, Puxeddu, & Testa Riva, 1999), where the major sublingual gland is located posteriorly and close to the submandibular gland (Leppi, 1967). The major sublingual gland, on the one hand, and the minor sublingual glands, on the other, have different embryogenic histories (Schulte & Von, 1913) and are drained by separate duct systems: the major sublingual gland by the duct of Bartholin, accompanying the submandibular duct, and each of the minor glands by a separate duct emptying directly into the mouth. The two types of sublingual gland are mixed glands with both mucous and seromucous cells. While the mucous cells in the minor sublingual glands clearly outnumber the seromucous cells, the mucous cells in the major sublingual gland just slightly outnumber the seromucous ones (Imai, Shibata, & Higashi, 1982; Riva et al., 1988). The parasympathetic cholinergic innervation of the parenchyma of salivary glands appears to be an invariable finding, whereas the sym- pathetic adrenergic innervation of the glands varies not only between species but also within the same species (Emmelin, 1967, 1981). In human minor sublingual, buccal and labial glands, Rossoni, Machado, and Machado (1979) found, as judged by cholinesterase histochemistry and catecholamine fluorescence, cholinergic innervation of the par- enchyma, while the adrenergic innervation of the parenchyma was virtually absent, despite being present around blood vessels. In contrast, the parenchyma of human parotid and submandibular glands displays a rich adrenergic innervation (Garrett, 1967; Norberg, Eneroth, & Hökfelt, 1970; Rossoni et al., 1979). No attention appears to have been paid to the human major salivary gland and its innervation in recent literature (see, for example, Ekström, Khosravani, Castagnola, & Messana, 2019; Proctor & Carpenter, 2014; Proctor, 2016; Sreebny & Vissink, 2010). Asakawa et al. (2015) recently reported the presence of neuronal nitric oxide synthase-positive nerves (used to indicate para- sympathetic nerves), as well as tyrosine hydroxylase-positive nerves https://doi.org/10.1016/j.archoralbio.2019.104571 Received 3 August 2019; Received in revised form 18 September 2019; Accepted 20 September 2019 Abbreviations: PBS, phosphate-buffered saline; ABC, avidin–biotin–peroxidase complex; TH, tyrosine hydroxylase; ChAT, choline acetyltransferase; DBH, dopamine- β-hydroxylase ⁎ Corresponding author. E-mail addresses: floy@unica.it (F. Loy), mpserra@unica.it (M.P. Serra), marianna.boi@unica.it (M. Boi), isola@unica.it (R. Isola), jorgen.ekstrom@pharm.gu.se (J. Ekström), quartu@unica.it (M. Quartu). Archives of Oral Biology 109 (2020) 104571 0003-9969/ © 2019 Elsevier Ltd. All rights reserved. T