Involvement of the neurosteroid 7α-hydroxypregnenolone in the courtship behavior of the male newt Cynops pyrrhogaster Fumiyo Toyoda a, ⁎, Itaru Hasunuma b , Tomoaki Nakada c , Shogo Haraguchi d , Kazuyoshi Tsutsui d , Sakae Kikuyama b, d a Physiology Department-I, Nara Medical University, Nara 634-8521, Japan b Department of Biology, Faculty of Science, Toho University, Chiba 274-8510, Japan c Department of Comparative and Behavior Medicine, Nippon Veterinary and Life Science University, Tokyo 180-8602, Japan d Department of Biology, Waseda University, and Center for Medical Life Science of Waseda University, Tokyo 162-8480, Japan abstract article info Article history: Received 13 March 2012 Revised 26 June 2012 Accepted 3 July 2012 Available online xxxx Keywords: Newt Courtship Behavior Neurosteroid 7α-hydroxypregnenolone Dopamine Reproductive behavior in amphibians, as in other vertebrate animals, is controlled by multiple hormones. A neurosteroid, 7α-hydroxypregnenolone, has recently been found to enhance locomotor activity in the male newt, Cynops pyrrhogaster. Here, we show that this neurosteroid is also involved in enhancing the expres- sion of courtship behavior. Intracerebroventricular (ICV) injection of 7α-hydroxypregnenolone enhanced courtship behavior dose-dependently in the sexually undeveloped males that had been pretreated with prolactin and gonadotropin, which is known to bring the males to a sexually developed state. But, unlike the case in the locomotion activity, 7α-hydroxypregnenolone did not elicit the behavior in males receiving no prior injections of these hormones. ICV administration of ketoconazole, an inhibitor of the steroidogenic enzyme cytochrome P450s, suppressed the spontaneously occurring courtship behavior in sexually active males. Supplementation with 7α-hydroxypregnenolone reversed the effect of ketoconazole in these ani- mals. It was also demonstrated that the effect of the neurosteroid on the courtship behavior was blocked by a dopamine D2-like, but not by a D1-like, receptor antagonist. These results indicate that endogenous 7α-hydroxypregnenolone enhances the expression of the male courtship behavior through a dopaminergic system mediated by a D2-like receptor in the brain. © 2012 Elsevier Inc. All rights reserved. Introduction During the breeding season, the male red-bellied newt, Cynops pyrrhogaster, attracts a female partner using the female-attracting pheromone sodefrin (Kikuyama et al., 1995). This pheromone is se- creted from the male's abdominal gland into the surrounding water and, through vigorous vibrations of the tail, the water is directed towards the female's snout. The male then creeps in front of the targeted female, who starts to follow the male, with her snout mak- ing contact with his tail. The male then discharges spermatophores, which are captured by the female's cloacal orifice and transported into the cloacal cavity (Kikuyama et al., 2003). Long-term research conducted by our group has demonstrated that multiple hormones, including androgen (Toyoda et al., 1993), prolactin (PRL) (Toyoda et al., 1993, 1996, 2005), gonadotropin (GTH) (Toyoda et al., 1993), and arginine vasotocin (AVT) (Toyoda et al., 2003), are involved either directly or indirectly in the expression of this behavior by the breed- ing red-bellied male newts. This is exemplified by the large increase in plasma concentrations of PRL (Matsuda et al., 1990), GTH (Tanaka et al., 1980), and androgen (Tanaka and Takikawa, 1983) in male newts during the breeding season in comparison to the non-breeding season. Various regions of the vertebrate brain are able to synthesize bioactive steroids from cholesterol (for reviews, see Baulieu, 1997; Compagnone and Mellon, 2000; Do Rego et al., 2009; Mellon and Vaudry, 2001; Tsutsui et al., 1999, 2003). Studies in mammals and various birds have revealed that these ‘brain-born’ steroids, termed neurosteroids, regulate various aspects of behavior activities, in- cluding aggressiveness, anxiety, sexual behavior, and locomotion (Matsunaga et al., 2004; Meieran et al., 2004; Melchior and Ritzmann, 1994; Tsutsui et al., 2008). In amphibians, Matsunaga et al. (2004) reported that a neurosteroid, 7α-hydroxypregnenolone, is present in abundance in the brain of the red-bellied newt. The synthesis of this neurosteroid was observed to reach a maximum level in the male newt during the spring breeding period concomi- tant with increases in their locomotor activity. Moreover, intra- cerebroventricular (ICV) injection of 7α-hydroxypregnenolone to non-breeding males caused an acute elevation of locomotor activity, an action shown to be mediated by dopamine via a dopamine D2-like receptor. Hormones and Behavior xxx (2012) xxx–xxx ⁎ Corresponding author at: Physiology Department-I, Nara Medical University, Shijo-chou 840, Kashihara, Nara 634-8521, Japan. Fax: +81 744 29 0306. E-mail address: tfumiyo@naramed-u.ac.jp (F. Toyoda). YHBEH-03414; No. of pages: 6; 4C: 0018-506X/$ – see front matter © 2012 Elsevier Inc. All rights reserved. doi:10.1016/j.yhbeh.2012.07.001 Contents lists available at SciVerse ScienceDirect Hormones and Behavior journal homepage: www.elsevier.com/locate/yhbeh Please cite this article as: Toyoda, F., et al., Involvement of the neurosteroid 7α-hydroxypregnenolone in the courtship behavior of the male newt Cynops pyrrhogaster, Horm. Behav. (2012), doi:10.1016/j.yhbeh.2012.07.001