Sex steroids control vertebrate behavior by modulating neural circuits specialized for sex steroid sensitivity. region that contains a crucial premotor song nucleus in vocal learning species. These results support the idea that AR signaling both centrally and peripherally is responsible for the activation of male manakin courtship and the arcopallium is likely a premotor site for AR-mediated displays site of PCRScript per manual (Stratagene) and clones were sequenced to confirm identity. We selected two clones pman200B and pman50B as templates for downstream in vitro transcription reactions (below) because they were identical except that the cDNA had ligated into the plasmid in opposite orientations. 2.3 Synthesis of riboprobe Antisense- and negative control sense-configured AR riboprobes were transcribed from two pmanAR clones after linearization with NotI (T7 RNA polymerase; Promega AZD8055 Madison WI). Riboprobes for ERα were synthesized by linearizing the plasmid containing the 2792-bp zebra finch AZD8055 ERα sequence (EJZER1 ) with MluI or EcoRI to obtain the antisense (T7) and sense (SP6 RNA polymerase) probes respectively. 33P labeled probes were prepared by in vitro AZD8055 transcription from ~100 ng of linearized plasmid 10 μl 33P-UTP (2000Ci/mmol; New England Nuclear Boston MA) and 1 μl of the appropriate RNA polymerase. Unincorporated nucleotides were removed with G-50 sephadex columns (Boehringer Manneheim Indianapolis IN). For in situ hybridization 20 μm tissue sections were processed as described in  with modifications: we did not include proteinase K digestion and hybridization was performed at 55 °C with 60 °C high-stringency post-hybridization washes. Dried sections were exposed to film (Kodak BioMax) for 2-3 days to estimate the length of exposure needed for subsequent emulsion autoradiography. The slides were then dipped in emulsion (Kodak NTB-2 Eastman Kodak Rochester NY) at 42 °C stored in light proof desiccated boxes at 4 °C and developed after 3-4 weeks (Eastman Kodak D-19; Fixer). Slides were examined under light and darkfield microscopy to determine the AZD8055 presence and distribution of labeled cells. 3 Results The neural distribution of AR ERα and their mRNAs in the brain has previously been described in details for several avian species [8 24 25 30 38 Thus here we focus only on the differences in AR and ERα mRNA expression between the GC-manakin and other studied bird species. In particular we found novel expression of AR mRNA in a large field of labeled cells in the arcopallium a pallial region that is the main output of the avian telencephalon and that in oscines contains the n. robustus arcopallii (RA) of the song system. To our knowledge this is the first report of substantial AR mRNA expression in the forebrain of a non-oscine bird . In the following description we adhere to the revised avian brain nomenclature . Although we studied only the distribution of the AR and ERα mRNAs hereafter we will speak of AR and ER expression for brevity. Previous studies have shown that the mRNA localization matches well the distribution of the receptor protein [8 24 25 30 38 3.1 Androgen receptors (AR) The distribution of AR in the GC-manakin forebrain resembled that previously described for non-oscine species in that no vocal control nuclei containing AR mRNA were found in the forebrain [5 24 38 However there was one noticeable exception: we found intense AR expression in the nucleus taeniae amygdalae (TnA) and in the arcopallium (Fig. 1D E G and H). Rostrally the field of AR expression extended from TnA to the arcopallium mediale and then laterally into the arcopallium dorsale following the structure previously called lamina archistriatilis dorsalis (LAD Fig. 1E). This field of AR expression extends caudally to occupy virtually the whole arcopallium Rabbit Polyclonal to FBLN2. intermedium (AI) extending from the medial end of the forebrain laterally to the LAD and from the caudal portion of TnA to the caudal end of the forebrain. This same region in oscines contains the AR-sensitive nucleus robustus arcopallii (RA) but a distinct RA is not recognizable in the GC-manakin (Fig. 1G and H). Fig. 1 Expression of AR-mRNA (panels (A)-(L)) and ERα-mRNA (panels (M)-(O)) in coronal sections of golden-collared manakin brain after in situ hybridization. Panels (A) (D) (G) (K) (M).