Coexistence of non-adrenergic non-cholinergic inhibitory and excitatory neurotransmitters in a large neuronal subpopulation in the vaginal segment of the chicken oviduct Anna Costagliola a , Joris G. De Man b , Mariusz Majewski c , Mirek Lakomy c , Aldo Cecio a , Patrick Robberecht d , Paul A. Pelckmans b , Dirk Adriaensen e , Jean-Pierre Timmermans e, * a Department of Biological Structures, Functions and Technology, Faculty of Veterinary Medicine-University Federico II, Naples, Italy b Division of Gastroenterology, Faculty of Medicine-University of Antwerp, Antwerp, Belgium c Department of Functional Morphology, Faculty of Veterinary Medicine Warmia and Mazury University of Olsztyn, Olsztyn, Poland d Department of Biochemistry, Faculty of Medicine-Universite ´ Libre de Bruxelles, Brussels, Belgium e Department of Biomedical Sciences, University of Antwerp, Laboratory of Cell Biology and Histology, Groenenborgerlaan 171, B-2020 Antwerpen, Belgium Received 26 March 2004; accepted 6 April 2004 Abstract The presence, distribution and smooth muscle motor effects of galanin and pituitary adenylate cyclase activating peptide (PACAP) were studied in the nerves of the vaginal part of the oviduct of egg-laying hens. Galanin and PACAP immunoreactivity were found both in neuronal perikarya and nerve fibres within the wall of the vaginal segment. Both populations showed a similar distribution pattern. Particularly the circular muscle and the intramural vascular net were richly innervated. A few galanin- and PACAP-IR nerve fibres extended up to the mucosal folds. Multiple labelling showed galanin to be colocalised with PACAP as well as with vasoactive intestinal polypeptide (VIP) and nitric oxide synthase (NOS) in a large, partly intrinsic neuronal subpopulation innervating the smooth muscle wall. Pharmacological in vitro experiments showed that isolated vaginal muscle strips had a spontaneous basal activity that was not affected by the neuronal conductance blocker tetrodotoxin (TTX). Galanin induced concentration-dependent contractions that were TTX-insensitive. PACAP, VIP, nitric oxide (NO) and the NO donor nitroglycerin caused concentration-dependent relaxations that were TTX-insensitive. Electrical field stimulation of isolated muscle strips induced frequency-dependent relaxations that were blocked by TTX and reduced by the NOS blocker L-nitroarginine. These data provide evidence that the vaginal part of the oviduct contains a largely intrinsic, neuronal subpopulation, capable of releasing multiple non-adrenergic, non-cholinergic (NANC) motor agents for the control of local muscular activities. In addition, we provided pharmacological evidence that VIP, NO and PACAP exert an inhibitory and galanin an excitatory action on isolated muscle strips of the vaginal part of the chicken oviduct. Our results suggest that these NANC neurotransmitters play an important role in the regulation of neuromuscular activity in this region. D 2004 Elsevier B.V. All rights reserved. Keywords: Chicken; Lower oviduct; Oviposition; Motility; Autonomic nervous system 1. Introduction The vaginal part of the chicken oviduct is a highly distensible muscular tube that allows the passage of the rigid shelled egg by a mechanism called oviposition. Func- tionally, when the egg is in the shell gland, the vagina is tonically closed to avoid premature oviposition. Subse- quently, the strong contraction of the smooth musculature of the shell gland forces the egg through the vagina, which relaxes to accomplish oviposition. An alteration in normal avian distal segment tone and motility may lead to prema- ture ovideposition (Lake and Gilbert, 1964), similarly to the abortion that can be observed in rat under experimental conditions (Doyle and Margolis, 1963). Oviposition occurs under neural control as well as under hormonal control, including the actions of arginine vasotocin (Rzasa and Ewy, 1970), which links specific receptors (Koike et al., 1988; Takahashi et al., 1992, 1998), and of ovarian prostaglandins (Wechsung and Houvenaghel, 1976). Sympathetic and para- sympathetic nerves reach the distal region via the large blood vessels (Gilbert and Lake, 1963). Noradrenergic nerve 1566-0702/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.autneu.2004.04.002 * Corresponding author. Tel.: +32-3-265-33-27; fax: +32-3-265-33-01. E-mail address: jean-pierre.timmermans@ua.ac.be (J.-P. Timmermans). www.elsevier.com/locate/autneu Autonomic Neuroscience: Basic and Clinical 112 (2004) 37– 48