Journal of Molecular Neuroscience 15 Volume 30, 2006 *Author to whom all correspondence and reprint requests should be addressed. E-mail: Katrin.S.Lips@anatomie. med.uni-giessen.de Coexpression and Spatial Association of Nicotinic Acetylcholine Receptor Subunits 7 and 10 in Rat Sympathetic Neurons Katrin Susanne Lips,* ,1 Peter König, 1 Katrin Schätzle, 1 Uwe Pfeil, 1 Gabriela Krasteva, 1 Markus Spies, 1 Rainer Viktor Haberberger, 1 Sergei A. Grando, 2 and Wolfgang Kummer 1 1 Institute for Anatomy and Cell Biology and University of Giessen Lung Center, Justus-Liebig-University, 35385 Giessen, Germany; and 2 Department of Dermatology, University of California at Davis, Sacramento, CA 95817 Introduction Fast excitatory synaptic transmission in sympa- thetic ganglia is mediated by nicotinic acetylcholine receptors (nAChRs). Although it is known that the nAChR 7-subunit occurs in sympathetic ganglia, the expression of the recently cloned subunit 10 (Elgoyhen et al., 2001; Lustig et al., 2001; Sgard et al., 2002) has not been analyzed. Until now, functional receptors containing 10-subunits have been found only in combination with 9-subunits (Elgoyhen et al., 2001; Lustig et al., 2001; Sgard et al., 2002). The 9-sub- unit exhibits a restricted expression pattern, whereas the 10-subunit is expressed more widely. This broad distribution resembles more closely that known for subunit 7 than for subunit 9. On this background, we investigated the distribution of nAChR subunits 7, 9, and 10 in rat sympathetic ganglia and stud- ied a possible interaction between subunit 7 and potential partners by double-labeling immunofluo- rescence and fluorescence resonance energy transfer (FRET) (Kam et al., 1995; Jares-Erijman and Jovin, 2003). Materials and Methods Experiments were performed on sympathetic ganglia of 30 adult Wistar rats. RT-PCR, in situ hybridization, Western blot, isolation of sympathetic Journal of Molecular Neuroscience Copyright © 2006 Humana Press Inc. All rights of any nature whatsoever are reserved. ISSN0895-8696/06/30:15–16/$30.00 JMN (Online)ISSN 1559-1166 DOI 10.1385/JMN/30:1-2:15 ORIGINAL ARTICLE neurons, and immunofluorescence were performed, as described previously (Lips et al., 2002; Haberberger et al., 2000). For FRET analysis double-labeling immunofluorescence was performed and analyzed using the FRET acceptor bleaching function of Leica- Confocal Software of the confocal laser scanning microscope (CLSM [TCSSP2 AOBS, Leica, Mannheim, Germany]). FRET efficiency was calculated as per- centage of increase of the donor fluorescence after photobleaching of the acceptor. Statistical testing was done by the Kruskal-Wallis test, followed by the Mann-Whitney test, using SPSS software, and p < 0.05 was set as level for significance. Results and Discussion Using RT-PCR, nonradioactive in situ hybridiza- tion, Western blotting, and double-labeling immuno- fluorescence, the 10-subunit was detected in noradrenergic and non-noradrenergic sympathetic neurons and in pericellular terminals predominantly around non-noradrenergic perikarya. The 9-subunit, which forms functional heteropentamers with the 10-subunit in hair cells, was not detected at protein level and only inconsistently and weakly at mRNA level. In contrast, 7-subunit mRNA was expressed by all postganglionic neurons, and 7-subunit protein