J. E. Rash et al.: Double-Replica FRIL Analysis of Gap Junctions imaged by LM in red ﬂuorescence (for neuronal markers) and green (for Cx43, Cx32 or Cx26), the overlay would have appeared to support Cx26 (or any other astrocyte connexin) between the two crossing neuronal processes. Thus, this image graphically demonstrates why ultrastructural approa- ches are essential for eliminating ambiguities of connexin assignment to speciﬁc cell types in CNS tissue. In addition to those early immunoﬂuorescence reports suggesting that neurons express multiple connexins that are now widely recognized to be ‘‘glial’’ [e.g., Cx26, Cx30, Cx32, Cx43 and Cx47 (Chang et al. 1999; Nadarajah et al. 1996, 1997; Nadarajah and Parnavelas 1999; Teubner et al. 2001; Venance et al. 2000; Zhang et al. 2000)], both Cx32 and Cx26 proteins were reported to occur between neurons at putative gap junctions, as identiﬁed by the presence of one to three silver-intensiﬁed gold beads at areas where membranes ‘‘tended to approach’’ (Fig. 2d, e); but these areas were not otherwise recognizable as gap junctions, even in these purported ‘‘consecutive serial sections.’’ Unaccountably, the contacting membranes had reversed contour in the successive sections, and the cytoplasm of one section was heavily stained for tyrosine hydroxylase/ peroxidase (Fig. 2d), whereas the successive section had little or no staining (Fig. 2e), suggesting that the samples may have been misidentiﬁed as representing consecutive serial sections. In contrast, FRIL (Fig. 2f) unambiguously revealed gap junctions as clusters of 10-nm P-face intra- membrane particles (IMPs) and/or 9-nm E-face pits (Goodenough and Revel 1970). [P-face = protoplasmic leaﬂet, E-face = extraplasmic leaﬂet; established nomen- clature deﬁned in Branton et al. (1975).] By FRIL, both neuronal and glial gap junctions were further conﬁrmed by labeling with multiple immunogold beads for appropriate cell type–speciﬁc connexins and only in the appropriate ultrastructurally identiﬁed cell types (Rash et al. 2001). To date, no neuronal gap junctions have been detected by FRIL that were immunogold-labeled for any of the consensus ‘‘glial’’ connexins. However, more than 3,000 neuronal gap junctions have been detected that were labeled for Cx36 (Kamasawa et al. 2006; Rash et al. 2005, 2007a, b), and *100 have been detected in rodent retina that were labeled for Cx45 (Li et al. 2008a) (see the fol- lowing); but none were labeled for glial connexins, either within neuronal hemiplaques or within the hemiplaques of neuronal coupling partners. This latter observation means that in normal CNS tissues neurons do not couple with glial cells, regardless of the connexins present in each. Pre- sumably, the neuronal connexins are nonpermissive with glial connexins. In contrast, in many of the same double- and triple-labeled samples, many thousands of glial gap junctions were cumulatively labeled by tens of thousands of gold beads for glial connexins, each gold bead repre- senting a separate conﬁrmation of the target connexin in 337 those gap junctions (Nagy et al. 2004; Rash et al. 2001). With no consensus glial connexins ever detected in ultra- structurally identiﬁed gap junctions of neurons and with thousands of glial gap junctions labeled for consensus glial connexins and never for neuronal connexins (Nagy et al. 2003, 2004; Nagy and Rash 2000; Rash et al. 2001), it is no longer appropriate to invoke those early reports as evidence for Cx26, Cx30, Cx32, Cx43 or Cx47 in neurons. On the other hand, Cx36 has been identiﬁed in sufﬁcient numbers of neuronal gap junctions and in sufﬁcient areas of the CNS to qualify as a reliable immunoﬂuorescence marker for electrical synapses in widespread brain regions [see com- panion paper (Lynn et al. 2012), this issue]. False Positives Arising from mRNA Detection Methods Even when combined with mRNA detection methods (e.g., in situ hybridization, RT-PCR or LacZ reporter methods), detection is common for multiple connexin mRNAs, including multiple glial connexin mRNAs in neurons (Chang et al. 1999; Venance et al. 2000; Zhang et al. 2000; Zhang 2010). In the decade since Fire et al. (1998) ﬁrst described mRNA suppression by microinter- fering RNA (miRNA), it has become clear that most classes of connexin mRNAs appear to be actively pre- vented from translation into protein by multiple miRNAs, which are particularly abundant in the mammalian brain (Bartel 2004; Berezikov et al. 2006; Farh et al. 2005; Krichevsky et al. 2003; Lim et al. 2005; Miska et al. 2004; Sempere et al. 2004), with some of the ‘‘seed matching sequences’’ for glial vs. neuronal connexins having been identiﬁed, consistent with active suppression of glial connexin mRNAs in neurons (Rash et al. 2005). We conclude that in CNS tissues there is such a high incidence of detection of diverse connexin mRNAs without detection of the corresponding connexin protein that such methods have been especially misleading in the identiﬁcation of the neuronal connexins that are actually expressed (i.e., false correlation of mRNA detection with protein detection). Speciﬁcally, one or two neuronal connexin mRNAs but at least ﬁve glial connexin mRNAs are routinely detected in neurons by single-cell RT-PCR and by in situ hybridization (Chang et al. 1999; Rash et al. 2005; Venance et al. 2000; Zhang et al. 2000; Zhang 2010). However, none of the ﬁve glial connexins that are routinely detected by these mRNA methods appear to be translated into proteins as none were detected by FRIL at ultrastructurally identiﬁed gap junctions, even in the same tissues and animal ages as examined by others (Li et al. 2008a; Rash et al. 2005, 2007a, b). To address these important issues of gap junction connexin composition and the ‘‘sidedness’’ of connexin expression, ultrastructural approaches have been employed, 123