1 Introduction Nagy et al., 2004 Belluardo et al., 2000; Condorelli et al., 2000; Rash et al., 2000 Deans et al., 2001 Hormuzdi et al., 2001; Buhl et al., 2003 Landisman et al., 2002 Long et al., 2002; De Zeeuw et al., 2003; Leznik and Llinas, 2005 Christie et al., 2005 Gogan et al., 1977; Walton and Navarrete, 1991; Chang et al., 1999; Mentis et al., 2002 Logan et al., 1996; Nolan et al., 1999 Marina et al., 2006; Tresch and Kiehn, 2000 Rash et al., 2000; Lee et al., 2005 2 Materials and methods 2.1 Tissue preparation − 1 Logan et al., 1996; Parenti et al., 2000 2.2 Immunohistochemistry Bittman et al., 2002 Marina et al., 2002 2.3 Image analysis The anatomical distribution of Cx36 immunostaining was determined by visual inspection through 63× and 100× objectives with an Olympus BH2-RFCA epifluorescence microscope equipped with light filters (exciter 380–490 nm, emitter 519 nm for the green channel). ChAT-Immunoreactive (ir) neurons were considered positive for Cx36 if punctate staining was observed within the cell body or along the dendritic processes. The cellular distribution of Cx36 in sympathetic preganglionic and somatic motoneurons was analyzed using a Leica TCS NT SP laser scanning confocal microscope with argon, krypton, and helium-neon lasers. Green and red channels were each scanned sequentially through a 63× objective. Alexa Fluor 488 labelling was observed by exciting at 488 nm and collecting at 515–550 nm. Alexa Fluor 568 labelling was visualized by using excitation at 568 nm and collecting at 590–626 nm. Serial optical sections were taken at intervals of 0.5 μm starting at the upper cell surface, i.e., the first optical section where cytoplasmic ChAT staining became visible, and all the way down through the cell to the lower cell surface, i.e., where ChAT staining was no longer detected. Composite figures were created for each optical section by merging both channels using Image J 1.37c (National Institutes of Health, USA). Colocalization of Cx36 (green) and cytoplasmic ChAT immunofluorescence (red) appeared as yellow. Thus, green puncta on the cell surface, i.e., surrounding ChAT-ir cytoplasm, suggested the presence of Cx36 on the plasma membrane; yellow intracellular puncta in ChAT-positive regions were indicative of cytoplasmic Cx36. Serial sections were projected as a single image to create a 2D reconstruction of 3D data set from the sections of the cell bodies and dendritic processes, using Leica Confocal Software (Leica Microsystems Heidelberg GmbH). Both the individual and projected images were saved directly to a computer as TIFF files. Images were minimally adjusted for brightness and contrast with Adobe Photoshop® 6 running on a Dell Pentium 4 PC. Images were then imported into Adobe illustrator® CS2 where groups of images were assembled and labelled. 3 Results 3.1 Connexin 36 immunostaining in the inferior olivary nucleus Fig. 1 Fig. 1 3.2 Connexin36 immunostaining in the spinal cord Rash et al., 2000; Lee et al., 2005 Fig. 2 3.3 Choline acetyltransferase immunostaining in the spinal cord Figs. 2 and 3 Cabot (1990; see Fig. 2A) Paxinos and Watson (1998; see Fig. 3) 3.4 Connexin 36 immunostaining in sympathetic preganglionic motoneurons Figs. 2 and 3 Fig. 2 Figs. 2 Fig. 2 Fig. 2 3.5 Connexin 36 immunostaining in somatic motoneurons Fig. 3 4 Discussion 4.1 Methodological considerations Rash et al., 2000; Placantonakis et al., 2006 Bittman et al., 2002 Placantonakis et al., 2006 Lee et al. (2005) Chang et al. (2000) Fig. 1 4.2 Cellular distribution of Cx36 immunostaining in ChAT-ir neurons Yeager et al., 1998; Martin and Evans, 2004 Yeager et al., 1998 Chang et al., 1999; Chang et al., 2000; Parenti et al., 2000 Becker and Navarrete, 1990; Shen and Dun, 1990; Mentis et al., 2002 van der Want et al., 1998 Matsumoto et al., 1988 Qin et al., 2005 Cabot et al., 1994 4.3 Physiological role of gap junctional communication between sympathetic preganglionic motoneurons Logan et al., 1996; Nolan et al., 1999 Logan et al., 1996 Nolan et al., 1999 Marina et al., 2006 4.4 Physiological role of gap junctional communication between somatic motoneurons Gogan et al., 1977; Becker and Navarrete, 1990; Walton and Navarrete, 1991; Chang et al., 1999; Mentis et al., 2002 Kiehn and Tresch, 2002 Becker and Navarrete 1990; Mentis et al., 2002 Gogan et al., 1977; Becker and Navarrete, 1990; Walton and Navarrete, 1991; Chang et al., 1999; Mentis et al., 2002 Chang et al., 1999 Chang et al., 1999 Lee et al., 2005 Chang et al., 1999 Bennett, 1997; Bennett and Zukin 2004 Mentis et al., 2002 5 Conclusions Rash et al., 2000; Lee et al., 2005