340 sections of adult rat replica were prepared by DR-SDS- FRL and simultaneously double-labeled for Cx36 and Cx45. We found more than 160 gap junctions labeled for Cx36, 12 of which also contained Cx45. Eleven of those were found in the matched complementary replica (Fig. 3c, c0), and all 11 of the complements also had Cx45, along with Cx36. None were found with Cx45 alone, and no additional Cx45-labeled gap junctions were found in the other complement. This means that of 11 examples of Cx45-containing gap junctions encountered, all contained both Cx36 and Cx45 on both sides, thereby demonstrating that 100 % of those 22 coupled cells synthesized both Cx45 and Cx36. Moreover, the two connexins appeared to reside in separate domains, with labeling for Cx45 aligned with (i.e., was opposite) labeling for Cx45 in the matched hemiplaque and labeling for Cx36 aligned with labeling for Cx36 (Fig. 3, matching inscribed quadrants). Thus, these gap junctions were not heterotypic, as proposed by others, nor did either side need to contain additional unidentiﬁed connexins for establishing permissive connexon channels. Rather, those 11 gap junctions were bihomotypic, with Cx45 apparently coupling to Cx45 and Cx36 apparently coupling to Cx36. This overcame the problem of what had previously appeared to be heterotypic coupling between nonpermissive Cx45 and Cx36. Finally, the fortuitous segregation of connexin labels within gap junctions (rather than labels being completely intermixed) was consistent with suggestions that the connexons were in homomeric domains (i.e., each domain within an individual gap junc- tion plaque contained only a single connexin type). DR-FRIL Revealed Heterotypic Gap Junctions at Synapses on Neurons of Goldﬁsh Hindbrain Neurons in the tetrapod lineage have Cx36 as their primary connexin. In contrast, teleost ﬁsh duplicated their entire genomes from the parent vertebrate lineage, resulting in two homologs of mammalian Cx36, which diverged slightly as Cx34.7 and Cx35 ([85 % homology). These connexins vary primarily in their phosphorylation sequen- ces [Cx34.7 lacks a phosphorylation site for CaMKII (Flores et al. 2010)] and in their membrane targeting sequences (O’Brien et al. 1998). In a ﬁrst step toward investigating the presence of these two homologs in gold- ﬁsh, we applied DR-FRIL to the analysis of gap junctions in a wide variety of neurons in the hindbrain. For comparison to our previous thin-section TEM ima- ges of heterotypic coupling at O:A gap junctions (Fig. 4a, inset), we show a rare cross-fractured gap junction with heterotypic labeling (Cx35 presynaptic and Cx34.7 post- synaptic, Fig. 4b) and matched DR complements of the an en face view of a gap junction on a reticulospinal neuron (Fig. 4c, d), with only small (5 nm) gold beads labeling 123 J. E. Rash et al.: Double-Replica FRIL Analysis of Gap Junctions postsynaptic connexins and only larger (10 nm) gold beads labeling presynaptic connexins. A diagram depicting the formation of a corresponding DR-FRIL replica is shown (Fig. 4e, f). These preliminary data document expression of Cx34.7 without Cx35 in hemiplaques of a goldﬁsh reticulospinal neuron, as well as Cx35 without Cx34.7 in the matching hemiplaques of its apposed axon terminal (Fig. 4c, d). In these matching complementary replicas, only Cx34.7 is detected in the reticulospinal neuron hemiplaque (Fig. 4c, arrowheads), whereas Cx35 is detected without Cx34.7 in the presynaptic hemiplaque that underlies the gap junction E-face pits (Fig. 4d). E-face pits are only faintly resolvable because of the 4- to 5-nm-thick carbon ‘‘precoat.’’ Although it is not yet determined whether the two types of coupled neurons (sensory and motor neurons) synthesize both connexins (with Cx35 targeted to axon terminals and Cx34.7 targeted to neuron soma and dendrites) or whether each neuronal subtype synthesizes only a single connexin isoform (Cx35 in sensory neurons and Cx34.7 in motor neurons), these data from DR-FRIL provide the molecular basis for a potential rectiﬁcation of electrical transmission between these neurons. Heterotypic gap junctions between neurons may con- tribute to the functional diversity of electrical transmission and provide a mechanism for preferred directionality of signaling (including electrical rectiﬁcation), while bi- and trihomotypic gap junctions may provide for bidirectional signaling but with the added property of differential mod- ulation of multiple conductance states for ions and small signaling molecules. Summary DR-FRIL and DR-SDS-FRL have unambiguously revealed individual gap junctions expressing two connexins—on the one hand, forming bihomotypic and, on the other hand, forming heterotypic junctions. As a now widely recognized type of synapse between neurons in the mammalian CNS, the conductance properties of electrical synapses and their regulation take on new importance. The two different connexin distributions demonstrated by DR-FRIL (i.e., bihomotypic vs. heterotypic) suggest the existence of pre- viously unrecognized mechanisms for regulating gap junction conductance states. Bi- and trihomotypic gap junctions are likely to increase the complexity and enhance the quality of synaptic communication provided by gap junctions. Likewise, heterotypic gap junctions provide not only for differential modulation on opposite sides of the same gap junction but also for the possibility of electrical rectiﬁcation in teleost CNS neurons. Because there appears so far to be only one connexin (Cx36) widely expressed in