334 expressed by cells in the central nervous system (CNS). Due to the cellular heterogeneity and morphological complexity of CNS tissue, assignment of connexin expression in, and understanding the formation of gap junctions between, particular cell types has been prob- lematic. Yet, this has become an important issue, especially in recent years by virtue of the identiﬁcation of several diseases with major neurological damage caused by mutations in connexins expressed in neural tissues. These include X-linked Charcot–Marie–Tooth disease (CMTX) (Bergoffen et al. 1993), resulting from mutations of Cx32; Pelizaeus–Merzbacher-like disease (PMLD), resulting from mutations of Cx47 (Tress et al. 2011; Uhlenberg et al. 2004), oculodentodigital dysplasia (ODDD), resulting from mutations of Cx43 (Paznekas et al. 2003); keratitis– ichthyosis–deafness syndrome, resulting from mutations of Cx26 (Kelsell et al. 1997; Melchionda et al. 2005); and childhood-onset myoclonic epilepsy, resulting from muta- tions of Cx36 in the noncoding region (Hempelmann et al. 2006; Mas et al. 2004). Understanding how these connexin mutations impact on physiological processes in the CNS and cause severe debilitating disease requires ﬁrm knowl- edge of the cell types expressing the mutated connexins, the subcellular and histological locations at which gap junctions may be disrupted by these abnormal connexins and the nature of the connexin coupling partners normally occurring at those locations. This knowledge acquired in studies of the glial connexins, as well as in studies of defective ion channels and water-transport pathways used for long-distance potassium siphoning and CNS water homeostasis, led to our formulation of the ‘‘gateway hypothesis’’ (Davidson and Rash 2011; Rash 2010) as a working paradigm for a generalized mechanism underlying the physiological and morphological aberrations found in CMTX, ODDD, PMLD, neuromyelitis optica, Alexander disease (van der Knaap et al. 2001) and other ‘‘leukodys- trophies’’ (white matter diseases). However, details of the molecular organization of glial gap junctions with their ﬁve J. E. Rash et al.: Double-Replica FRIL Analysis of Gap Junctions gap junction-forming connexins, as well as of gap junctions forming electrical synapses in the CNS, is a work in pro- gress. Here, we outline some outstanding difﬁculties and present a new approach that may help to resolve some of the existing limitations. Connexon Coupling Patterns Individual cell types express from one to four different connexins, allowing for the potential formation of ‘‘heter- omeric’’ connexons composed of two or more connexins, for which evidence has been obtained in only a few tissues in vivo (Jiang and Goodenough 1996; Sosinsky 1995). Some neurons express only a single connexin isoform (e.g., most express only Cx36), and these often form gap junc- tions with other neurons singly expressing the same connexin, thereby forming ‘‘homomeric’’ connexins in both cells that link to form ‘‘homotypic’’ intercellular channels (Fig. 1a, showing the simplest type of neuron-to- neuron gap junction, composed of Cx36, only). However, multiple connexin isoforms are expressed in many types of cells, providing for the possible formation of ‘‘bihomo- typic’’ and ‘‘trihomotypic’’ gap junctions. For example, astrocyte-to-astrocyte (A:A) gap junctions usually consist of two or three types of homotypic channels, forming, as one example, a Cx43:Cx43 plus Cx30:Cx30 plus Cx26:Cx26 trihomotypic gap junction (Fig. 1b). In addi- tion, when astrocytes (A) couple to oligodendrocytes (O), which express two other gap junction-forming connexins, Cx47 and Cx32, these ‘‘heterologous’’ A:O gap junctions, are necessarily ‘‘bi-’’ or ‘‘triheterotypic’’ (or even multi- heterotypic), composed of several combinations of ‘‘per- missive’’ coupling partners, three of which are illustrated in Fig. 1c. [Cx29 is also expressed in oligodendrocytes but does not form gap junctions (Altevogt and Paul 2004).] In A:O junctions, a distinct set of connexins on the astrocyte side (i.e., Cx26, Cx30, Cx43) link with a different set of Fig. 1 Generalized models of different connexin coupling patterns in the most common type of gap junctions in the CNS. a ‘‘Homotypic’’ neuronal gap junction, with intercellular channels composed of Cx36 coupling with Cx36. b ‘‘Trihomotypic’’ astrocyte-to-astrocyte gap junction, with intercellular channels composed of Cx43 coupling with Cx43, Cx30 coupling with Cx30 and Cx26 coupling with Cx26. 123 c ‘‘Triheterotypic’’ astrocyte-to-oligodendrocyte gap junction, with astrocyte Cx43 coupling with oligodendrocyte Cx47, astrocyte Cx30 coupling with oligodendrocyte Cx32 and astrocyte Cx26 coupling with oligodendrocyte Cx32. Additional permissive coupling pairs are discussed in the text