Functional autoradiography and gene expression analysis applied to the characterization of the a 2 -adrenergic system in the chicken brain Rebeca Diez-Alarcia a,b , Ricardo Mostany c , Severiano Dos-Anjos d , Arsenio Ferna ´ ndez-Lo ´ pez d, * a Centro de Investigacio ´n Biome ´dica en Red de Salud Mental, CIBERSAM, Spain b Departamento de Farmacologı´a, Universidad del Paı´s Vasco (UPV/EHU), Leioa, Vizcaya, Spain c Departamento de Fisiologı´a y Farmacologı´a, Facultad de Medicina, Universidad de Cantabria, Santander, Spain d A ´ rea de Biologı´a Celular, Instituto de Biomedicina, Universidad de Leo ´n, Leo ´n, Spain 1. Introduction Avian biological diversity and its complex social behaviour make an excellent model for studying neuronal functions such as seasonal neurogenesis, apoptosis, learning, neuronal proliferation and plasticity (Bottjer and Arnold, 1997; Tramontin and Brenowitz, 2000). In previous studies we determined the pharmacological profile and distribution of the a 2 -adrenoceptor subtypes in the chicken central nervous system (CNS), showing that they are highly conserved receptors (Fernandez-Lopez et al., 1990, 1997) and confirming the presence of the three different a 2 -adreno- ceptor subtypes described for the human brain: a 2A -, a 2B -, and a 2C -adrenoceptors (Diez-Alarcia et al., 2006). Those data also showed a similar pharmacological profile between chicken and rat a 2 -adrenoceptors in the brain, and a highly conserved anatomical distribution of these receptors in birds and mammals CNS. Although chicken genes codifying for a 2 -adrenoceptors have not yet been sequenced, the comparison of primary and secondary structures of a 2 -adrenoceptors cloned for different species of mammals and fish shows a high correlation coefficient (Svensson et al., 1993; Ruuskanen et al., 2005). Now, in an attempt to better characterize the a 2 -adrenoceptor system in this species, we setup for the first time the [ 35 S]GTPgS autoradiography for a 2 - adrenoceptors in chicken brain tissue sections. Moreover, we also report the expression levels of the three a 2 -adrenoceptor subtypes determined by real-time RT-PCR. As members of the G protein-coupled receptors (GPCRs) family, the a 2 -adrenoceptors interaction with heterotrimeric G proteins Journal of Chemical Neuroanatomy 38 (2009) 282–291 ARTICLE INFO Article history: Received 22 April 2009 Received in revised form 8 September 2009 Accepted 9 September 2009 Available online 19 September 2009 Keywords: a 2 -Adrenoceptors Adenosine receptor Tissue section RT-PCR ABSTRACT Here we report a functional autoradiographic study of [ 35 S]GTPgS binding induced by a 2 -adrenoceptor activation in chicken brain tissue sections using both 10 4 M UK 14304 (bromoxidine or brimonidine) and 10 6 M epinephrine as a 2 -adrenoceptor agonists. Assays were performed using two different incubation buffers: glycylglycine or Tris–HCl. Changes in the [ 35 S]GTPgS basal binding values were detected, and different [ 35 S]GTPgS specific binding values were also obtained depending on the buffer used for each drug. The best results were obtained with epinephrine in Tris–HCl, with slightly higher stimulation values than the observed with UK 14304 in glycylglycine buffer. The effect of the addition of adenosine deaminase to the incubation buffer was also tested. This effect decreasing basal binding in chicken was very small when compared to mammals, according with differences found in adenosine 1 receptor expression levels. Structures presenting a 2 -adrenoceptor-mediated G i/o protein stimulation fitted with areas previously described as enriched in a 2 -adrenoceptors in chicken brain, and their homologous areas in mammals. These data confirm the specificity of the results and reinforce the implication of the a 2 -adrenoceptors in the function of these brain nuclei. On the other hand, the expression level of the different a 2 -adrenoceptor subtypes was tested with real-time PCR. Contrasting with the a 2 -adrenoceptor subtype distribution previously described with radioligand competition assays, where a 2A was the predominant a 2 -adrenoceptor subtype (75%); in the present work, the ratio of a 2A :a 2B/C gene expression was lower than expected both in telencephalon, tectum opticum, and cerebellum. ß 2009 Published by Elsevier B.V. Abbreviations: GPCR, G protein-coupled receptor; CNS, central nervous system; [ 35 S]GTPgS, [ 35 S]guanylyl-5 0 -O-(g-thio)-triphosphate; ADA, adenosine deaminase; E, epinephrine; ASB, agonist stimulated binding; NSB, non-specific binding; A1R, adenosine A1 receptor. * Corresponding author at: Departamento de Biologı ´a Molecular, Facultad de Ciencias Biolo ´ gicas y Ambientales, Universidad de Leo ´ n, Campus de Vegazana s/n, C.P. 24071, Leo ´ n, Spain. Tel.: +34 987 291485; fax: +34 987 291226. E-mail addresses: arsenio.fernandez@unileon.es, rdiezalarcia@gmail.com (A. Ferna ´ ndez-Lo ´ pez). Contents lists available at ScienceDirect Journal of Chemical Neuroanatomy journal homepage: www.elsevier.com/locate/jchemneu 0891-0618/$ – see front matter ß 2009 Published by Elsevier B.V. doi:10.1016/j.jchemneu.2009.09.002