Confocal imaging and phylogenetic considerations of the subcutaneous neurons in the Atlantic hagfish Myxine glutinosa Giacomo Zaccone, 1 Douglas S. Fudge, 2 Timothy M. Winegard, 2 Gioele Capillo, 1 Michal Kuciel, 3 Kengo Funakoshi 4 and Eugenia Rita Lauriano 1 1 Department of Environmental Science, Territory, Food and Health Security (S.A.S.T.A.S.), Viale Stagno d’Alcontres 31, I-98166, Messina, Italy; 2 Department of Integrative Biology, University of Guelph, Guelph, ON, N1G-2W1, Canada; 3 Department of Comparative Anatomy, Institute of Zoology, Jagiellonian University, Gronostajowa 9, 30-387, Krakow, Poland; 4 Department of Neuro- anatomy, Yokohama City University School of Medicine, 22-2 Seto, Kanazawa Ward, Yokohama, Kanagawa Prefecture 236-0027, Japan Keywords: subcutaneous neurons, slime glands, striated muscle, confocal immunohistochemistry, hagfish, phylogeny Accepted for publication: 19 December 2013 Abstract Zaccone, G., Fudge, D.S., Winegard, T.M., Capillo, G., Kuciel, M., Funakoshi, K. and Lauriano, E.R. 2014. Confocal imaging and phylogenetic considerations of the subcutaneous neurons in the Atlantic hagfish Myxine glutinosa.— Acta Zoologica (Stockholm) 00: 000–000. We used confocal microscopy and immunohistochemistry to characterize the morphology of the subcutaneous neurons and the innervation of the slime glands and striated muscles in the hagfish Myxine glutinosa. A rich plexus of 5HT-, ChAT- and TH-positive neurons is described in the capsule of the slime glands. These neurons, like those of the subcutaneous plexus, receive pericellular terminations from the axons of central cells. Capsular neurons receive innervation from 5HT-positive and nNOS-positive nerve fibres. Other nerve endings belonging to two separate nerve populations are identified in the striated muscles. They contain TH and nNOS immunoreactivity. Due to the lack of any topographical labelling, the cell origin and the projections of the neurons into the cranial and spinal nerves are unknown. This study pro- vides anatomical evidence of multiple (5HT and nNOS) peripheral innerva- tion of the neurons. However, it does not provide information about the function of these neurons in the hagfish. We suggest that hagfish neurons have a phylogenetic relationship with the spinal group of the dorsal cells of lam- preys and the supramedullary cells of teleosts. Giacomo Zaccone, Department of Environmental Science, Territory, Food and Health Security (S.A.S.T.A.S.), Viale Stagno d’Alcontres 31, I-98166 Messina, Italy. E-mails: gzaccone@unime.it; gioelecapilo@hotmail.it; elauriano@unime.it Introduction Cyclostomata comprise the living jawless fishes, hagfishes and lampreys. Morphological and molecular studies indicate that hagfishes and lampreys are sister groups, the Cyclostomata itself being a sister group to the vertebrates. Within Cyclosto- mata, hagfishes are less evolved than lampreys and constitute the most primitive craniates available for study (Forey and Janvier 1993; Mallat et al. 2001; Zardova and Meyer 2001). In the myotomes of hagfishes, two muscle fibre types are arranged in a characteristic sandwich pattern. The two fibre types differ in size, myofibrillar array, blood supply and inner- vation (Bone 1989). In gnathostomatous fish, the sympathetic chains are generally well developed, their arrangement resem- bling that found in the tetrapods. A unique feature in teleosts and holosteans is that the cephalic portions of the sympathetic chains that continue into the head bear ganglia in connection with the glossopharyngeal and vagus nerves. The connections between the ganglia and the cranial nerves are solely com- posed of postganglionic fibres (grey rami communicantes) (Kapoor and Khanna 2004). An interesting and puzzling feature of the innervation of myotomes in Cyclostomata is that lampreys have a dual innervation of muscle fibres whereas hagfishes are innervated by a single axon type. Hagfishes and lampreys differ in mus- cle innervation and in the shape of the muscle fibres, the © 2014 The Royal Swedish Academy of Sciences 1 Acta Zoologica (Stockholm) doi: 10.1111/azo.12068