NEUROSYSTEMS Ablation of connexin30 in transgenic mice alters expression patterns of connexin26 and connexin32 in glial cells and leptomeninges B. D. Lynn, 1 O. Tress, 2 D. May, 2 K. Willecke 2 and J. I. Nagy 1 1 Department of Physiology, Faculty of Medicine, University of Manitoba, Winnipeg, Manitoba, Canada 2 Institute of Genetics, Division of Molecular Genetics, University of Bonn, Bonn, Germany Keywords: astrocytes, connexin43, connexin47, gap junctions, oligodendrocytes Abstract Expression of connexin26 (Cx26), Cx30 and Cx43 in astrocytes and expression of Cx29, Cx32 and Cx47 in oligodendrocytes of adult rodent brain has been well documented, as has the interdependence of connexin expression patterns of macroglial cells in Cx32- and Cx47-knockout mice. To investigate this interdependence further, we examined immunofluorescence labelling of glial connexins in transgenic Cx30 null mice. Ablation of astrocytic Cx30, confirmed by the absence of immunolabelling for this connexin in all brain regions, resulted in the loss of its coupling partner Cx32 on the oligodendrocyte side of astrocyte–oligodendrocyte (A ⁄ O) gap junctions, but had no effect on the localization of astrocytic Cx43 and oligodendrocytic Cx47 at these junctions or on the distribution of Cx32 along myelinated fibres. Surprisingly, gene deletion of Cx30 led to the near total elimination of immunofluorescence labelling for Cx26 in all leptomeningeal tissues covering brain surfaces as well as in astrocytes of brain parenchyma. Moreover northern blot analysis revealed downregulation of Cx26 mRNA in Cx30-knockout brains. Our results support earlier observations on the interdependency of Cx30 ⁄ Cx32 targeting to A ⁄ O gap junctions and further suggest that Cx26 mRNA expression is affected by Cx30 gene expression. In addition, Cx30 protein may be required for co-stabilization of gap junctions or for co-trafficking in cells. Introduction Cells in the rodent central nervous system (CNS) collectively express over a dozen of the 20-member gap junction–connexin family of proteins that form intercellular communicating channels at appositions between cellular plasma membranes. In the past decade, it has become clear that different cell types in neural tissue express different connexins, leading to the concept of ‘cell-specific connexin expres- sion’, as originally proposed in studies of connexin localization in brain (Rash et al., 2001a,b). Thus, in adult rodent brain, neurons express predominately connexin36 (Cx36), astrocytes express Cx26, Cx30 and Cx43, oligodendrocytes express Cx29, Cx32 and Cx47, and leptomen- ingeal cells express Cx26, Cx30 and Cx43 (Rash et al., 2001a,b; Nagy et al., 2004, 2011; Rash, 2010). Among macroglial cells, Cx29 remains enigmatic because it is unable to form gap junctions (Ahn et al., 2008), while the remaining connexins contribute to a complex network or syncytium of cells linked by astrocyte–astrocyte (A ⁄ A) gap junctions and by astrocyte–oligodendrocyte (A ⁄ O) gap junctions. Recent dye- transfer experiments revealed functional inter-oligodendrocytic cou- pling (O ⁄ O) in the corpus callosum (Maglione et al., 2010), which was absent in the cerebral cortex (Wasseff & Scherer, 2011). Why these cells require multiple connexins for normal function is not yet clear, but may to some extent be related to their utilization of some of these connexins at different subcellular compartments (Mugnaini, 1986; Yamamoto et al., 1990; Wolburg & Rohlmann, 1995; Rash et al., 1997, 2001a,b; Kamasawa et al., 2005; Theis et al., 2005; Orthmann- Murphy et al., 2008; Rash, 2010). As not all connexins can form functional communicating channels with each other, an equally pressing question relates to the organization of connexin coupling partners at gap junctions between macroglial cells. For example, A ⁄ A gap junctions can consist of homotypic and ⁄ or heterotypic combina- tions of apposing connexins, while A ⁄ O gap junctions are necessarily heterotypic, meaning that a set of different connexins on the astrocyte side (i.e. Cx26, Cx30, Cx43) link with a different set of connexins on the oligodendrocyte side (i.e. Cx32, Cx47) (Scherer et al., 1995; Li et al., 1997; Nagy & Rash, 2000; Altevogt et al., 2002; Altevogt & Paul, 2004; Kleopa et al., 2004; Nagy et al., 2004). Recent studies of coupling permissiveness in N2A cells expressing glial connexins indicate that, in addition to permissive homotypic combinations (e.g. Cx30 ⁄ Cx30, Cx43 ⁄ Cx43, Cx32 ⁄ Cx32 and Cx47 ⁄ Cx47), functional coupling can occur between Cx30 ⁄ Cx32, Cx43 ⁄ Cx47 (Orthmann- Murphy et al., 2007b) and Cx30 ⁄ Cx47 (Magnotti et al., 2011). The likelihood that similar homotypic and heterotypic connexin pairing partners occur in vivo at A ⁄ A gap junctions and A ⁄ O gap junctions was supported by observations of selective interdependency of connexin targeting to glial junctions in transgenic mice. Ablation of oligodendrocytic Cx32 led to the loss of its presumed astrocytic Cx30 Correspondence: Dr J. I. Nagy, as above. E-mail: nagyji@ms.umanitoba.ca Received 25 April 2011, revised 2 September 2011, accepted 11 September 2011 European Journal of Neuroscience, Vol. 34, pp. 1783–1793, 2011 doi:10.1111/j.1460-9568.2011.07900.x ª 2011 The Authors. European Journal of Neuroscience ª 2011 Federation of European Neuroscience Societies and Blackwell Publishing Ltd European Journal of Neuroscience