The avian song control system is an excellent model in which to study the effects of gonadal steroid hormones on neural and behavioral plasticity. Several of the brain regions that control song behavior concentrate androgens andlor estrogens. Investigations of the distribution and regulation of androgen receptors have been limited by the lack of a reliable immunocytochemical method to detect androgen receptors in the songbird brain. We describe a protocol by which the PG-21 polyclonal antibody to the rat androgen receptor can be used to label androgen receptor-containing cells in the songbird brain. By treating songbirds of several species with testosterone 90 min before sauifce and by using relatively
IntroductionThe neural circuit that regulates song behavior in songbirds has emerged as a leading model for studying the effects of gonadal steroid hormones on brain structure and function. Song is a learned behavior that is sexually dimorphic in most species. The morphology of the brain nuclei that control song differs between sexes and between seasons (reviewed in Brenowitt and Kroodsma, in press;Arnold, 1992;DeVoogd, 1991;Konishi, 1989;Nottebohm, 1987).Both the organization and the activation of the song control system are strongly influenced by gonadal steroid hormones. Synergistic interactions of androgens and estrogens are essential for development of song control circuits in the brain and for production of song (reviewed in Brenowitz and K " a , in press). Song learning is influenced by gonadal steroids (Korsia and Bottjer, 1991;Marler et al., 1988). These hormones also play an important role in mediating seasonal plasticity of song nuclei in adult songbirds (Smith et al., 1993).Steroid autoradiography has been the primary tool used to study the distribution and regulation of gonadal steroid receptors in the song control system (Arnold et al., 1976;Zigmond et al., 1973). Autoradiographic studies of zebra finches (Zeniopygiaguttuta) and canaries (Serinur canariu) using [3H]-dihydrotestosterone (DHT) as a ligand indicate that androgen receptor-containing (AR+ ) cells are present in five song nuclei: the higher vocal center (HVC), the magnocellular nucleus of the anterior neostriatum (MAN), the robust nucleus of the archistriatum (RA), the intercollicular nucleus (ICo), and the tracheosyringeal portion of the hypoglossal nucleus (nXIIts) (Gahr, 1990;Nordeen et al., 1986). Androgen accumulation is also observed in the preoptic area, the magnocellular paraventricular nucleus, and the tuberal nucleus in the hypothalamus of these species '(Gahr, 1990; Nordeeti et al., 1986).The autoradiographic technique has several limitations as a means of studying the distribution of cells that contain AR. With 3H as the radioactive isotope, brain sections must be exposed to emulsion for weeks to months before adequate numbers of silver grains are reduced in the emulsion to allow discrimination of labeled cells. 0-Particles emitted by the decay of 3H penetrate poorly through more than 3 pm of tissue (Clark et al., 1990;Arnold, ...