Kimberly G. V. Davidson

Aquaporin-4 square array assembly: Opposing actions of M1 and M23 isoforms

Osmotic homeostasis in the brain involves movement of water through aquaporin-4 (AQP4) membrane channels. Perivascular astrocyte end-feet contain distinctive orthogonal lattices (square arrays) assembled from 4- to 6-nm intramembrane particles (IMPs) corresponding to individual AQP4 tetramers. Two isoforms of AQP4 result from translation initiation at methionine residues M1 and M23, but no functional differences are known. In this study, Chinese hamster ovary cells were transfected with M1, M23, or M1+M23 isoforms, and AQP4 expression was confirmed by immunoblotting, immunocytochemistry, and immunogold labeling. Square array organization was examined by freeze-fracture electron microscopy. In astrocyte end-feet, >90% of 4- to 6-nm IMPs were found in square arrays, with 65% in arrays of 13-30 IMPs. In cells transfected with M23, 95% of 4- to 6-nm IMPs were in large assemblies (rafts), 85% of which contained >100 IMPs. However, in M1 cells, >95% of 4- to 6-nm IMPs were present as singlets, with <5% in incipient arrays of 2-12 IMPs. In M1+M23 cells, 4- to 6-nm IMPs were in arrays of intermediate sizes, resembling square arrays in astrocytes. Structural cross-bridges of 1 × 2 nm linked >90% of IMPs in M23 arrays (≈1,000 cross-bridges per μm2) but were rarely seen in M1 cells. These studies show that M23 and M1 isoforms have opposing effects on intramembrane organization of AQP4: M23 forms large square arrays with abundant cross-bridges; M1 restricts square array assembly.

Identification of Cells Expressing Cx43, Cx30, Cx26, Cx32 and Cx36 in Gap Junctions of Rat Brain and Spinal Cord

We have identified cells expressing Cx26, Cx30, Cx32, Cx36 and Cx43 in gap junctions of rat central nervous system (CNS) using confocal light microscopic immunocytochemistry and freeze-fracture replica immunogold labeling (FRIL). Confocal microscopy was used to assess general distributions of connexins, whereas the 100-fold higher resolution of FRIL allowed co-localization of several different connexins within individual ultrastructurally-defined gap junction plaques in ultrastructurally and immunologically identified cell types. In >4000 labeled gap junctions found in >370 FRIL replicas of gray matter in adult rats, Cx26, Cx30 and Cx43 were found only in astrocyte gap junctions; Cx32 was only in oligodendrocytes, and Cx36 was only in neurons. Moreover, Cx26, Cx30 and Cx43 were co-localized in most astrocyte gap junctions. Oligodendrocytes shared intercellular gap junctions only with astrocytes, and these heterologous junctions had Cx32 on the oligodendrocyte side and Cx26, Cx30 and Cx43 on the astrocyte side. In 4 and 18 day postnatal rat spinal cord, neuronal gap junctions contained Cx36, whereas Cx26 was present in leptomenigeal gap junctions. Thus, in adult rat CNS, neurons and glia express different connexins, with “permissive” connexin pairing combinations apparently defining separate pathways for neuronal vs. glial gap junctional communication.

Temporary loss of perivascular aquaporin-4 in neocortex after transient middle cerebral artery occlusion in mice

The aquaporin-4 (AQP4) pool in the perivascular astrocyte membranes has been shown to be critically involved in the formation and dissolution of brain edema. Cerebral edema is a major cause of morbidity and mortality in stroke. It is therefore essential to know whether the perivascular pool of AQP4 is up- or down-regulated after an ischemic insult, because such changes would determine the time course of edema formation. Here we demonstrate by quantitative immunogold cytochemistry that the ischemic striatum and neocortex show distinct patterns of AQP4 expression in the reperfusion phase after 90 min of middle cerebral artery occlusion. The striatal core displays a loss of perivascular AQP4 at 24 hr of reperfusion with no sign of subsequent recovery. The most affected part of the cortex also exhibits loss of perivascular AQP4. This loss is of magnitude similar to that of the striatal core, but it shows a partial recovery toward 72 hr of reperfusion. By freeze fracture we show that the loss of perivascular AQP4 is associated with the disappearance of the square lattices of particles that normally are distinct features of the perivascular astrocyte membrane. The cortical border zone differs from the central part of the ischemic lesion by showing no loss of perivascular AQP4 at 24 hr of reperfusion but rather a slight increase. These data indicate that the size of the AQP4 pool that controls the exchange of fluid between brain and blood during edema formation and dissolution is subject to large and region-specific changes in the reperfusion phase.

Gap junctions on hippocampal mossy fiber axons demonstrated by thin-section electron microscopy and freeze–fracture replica immunogold labeling

Gap junctions have been postulated to exist between the axons of excitatory cortical neurons based on electrophysiological, modeling, and dye-coupling data. Here, we provide ultrastructural evidence for axoaxonic gap junctions in dentate granule cells. Using combined confocal laser scanning microscopy, thin-section transmission electron microscopy, and grid-mapped freeze–fracture replica immunogold labeling, 10 close appositions revealing axoaxonic gap junctions (≈30–70 nm in diameter) were found between pairs of mossy fiber axons (≈100–200 nm in diameter) in the stratum lucidum of the CA3b field of the rat ventral hippocampus, and one axonal gap junction (≈100 connexons) was found on a mossy fiber axon in the CA3c field of the rat dorsal hippocampus. Immunogold labeling with two sizes of gold beads revealed that connexin36 was present in that axonal gap junction. These ultrastructural data support computer modeling and in vitro electrophysiological data suggesting that axoaxonic gap junctions play an important role in the generation of very fast (>70 Hz) network oscillations and in the hypersynchronous electrical activity of epilepsy.

Connexin47, connexin29 and connexin32 co-expression in oligodendrocytes and cx47 association with zonula occludens-1 (zo-1) in mouse brain

Gap junctions between glial cells in mammalian CNS are known to contain several connexins (Cx), including Cx26, Cx30 and Cx43 at astrocyte-to-astrocyte junctions, and Cx29 and Cx32 on the oligodendrocyte side of astrocyte-to-oligodendrocyte junctions. Recent reports indicating that oligodendrocytes also express Cx47 prompted the present studies of Cx47 localization and relationships to other glial connexins in mouse CNS. In view of the increasing number of connexins reported to interact directly with the scaffolding protein zonula occludens-1 (ZO-1), we investigated ZO-1 expression and Cx47/ZO-1 interaction capabilities in brain, spinal cord and Cx47-transfected HeLa cells. From counts of over 9000 oligodendrocytes labeled by immunofluorescence in various brain regions, virtually all of these cells were found to express Cx29, Cx32 and Cx47. Oligodendrocyte somata displayed robust Cx47-immunopositive puncta that were co-localized with punctate labeling for Cx32 and Cx43. By freeze-fracture replica immunogold labeling, Cx47 was abundant on the oligodendrocyte-side of oligodendrocyte/astrocyte gap junctions. By immunofluorescence, labeling for Cx47 along myelinated fibers was sparse in most brain regions, whereas Cx29 and Cx32 were previously found to be concentrated along these fibers. By immunogold labeling, Cx47 was found in numerous small gap junctions linking myelin to astrocytes, but not within deeper layers of myelin. Brain subcellular fractionation revealed a lack of Cx47 enrichment in myelin fractions, which nevertheless contained an enrichment of Cx32 and Cx29. Oligodendrocytes were immunopositive for ZO-1, and displayed almost total Cx47/ZO-1 co-localization. ZO-1 was found to co-immunoprecipitate with Cx47, and pull-down assays indicated binding of Cx47 to the second PDZ domain of ZO-1. Our results indicate widespread expression of Cx47 by oligodendrocytes, but with a distribution pattern in relative levels inverse to the abundance of Cx29 in myelin and paucity of Cx29 in oligodendrocyte somata. Further, our findings suggest a scaffolding and/or regulatory role of ZO-1 at the oligodendrocyte side of astrocyte-to-oligodendrocyte gap junctions.

Connexin45-Containing Neuronal Gap Junctions in Rodent Retina Also Contain Connexin36 in Both Apposing Hemiplaques, Forming Bihomotypic Gap Junctions, with Scaffolding Contributed by Zonula Occludens-1

Mammalian retinas contain abundant neuronal gap junctions, particularly in the inner plexiform layer (IPL), where the two principal neuronal connexin proteins are Cx36 and Cx45. Currently undetermined are coupling relationships between these connexins and whether both are expressed together or separately in a neuronal subtype-specific manner. Although Cx45-expressing neurons strongly couple with Cx36-expressing neurons, possibly via heterotypic gap junctions, Cx45 and Cx36 failed to form functional heterotypic channels in vitro. We now show that Cx36 and Cx45 coexpressed in HeLa cells were colocalized in immunofluorescent puncta between contacting cells, demonstrating targeting/scaffolding competence for both connexins in vitro. However, Cx36 and Cx45 expressed separately did not form immunofluorescent puncta containing both connexins, supporting lack of heterotypic coupling competence. In IPL, 87% of Cx45-immunofluorescent puncta were colocalized with Cx36, supporting either widespread heterotypic coupling or bihomotypic coupling. Ultrastructurally, Cx45 was detected in 9% of IPL gap junction hemiplaques, 90–100% of which also contained Cx36, demonstrating connexin coexpression and cotargeting in virtually all IPL neurons that express Cx45. Moreover, double replicas revealed both connexins in separate domains mirrored on both sides of matched hemiplaques. With previous evidence that Cx36 interacts with PDZ1 domain of zonula occludens-1 (ZO-1), we show that Cx45 interacts with PDZ2 domain of ZO-1, and that Cx36, Cx45, and ZO-1 coimmunoprecipitate, suggesting that ZO-1 provides for coscaffolding of Cx45 with Cx36. These data document that in Cx45-expressing neurons of IPL, Cx45 is almost always accompanied by Cx36, forming “bihomotypic” gap junctions, with Cx45 structurally coupling to Cx45 and Cx36 coupling to Cx36.

Connexin32-Containing Gap Junctions in Schwann Cells at the Internodal Zone of Partial Myelin Compaction and in Schmidt–Lanterman Incisures

In vertebrate peripheral nerves, the insulating myelin sheath is formed by Schwann cells, which generate flattened membrane processes that spiral around axons and form compact myelin by extrusion of cytoplasm and adhesion of apposed intracellular and extracellular membrane surfaces. Cytoplasm remains within the innermost and outermost tongues, in the paranodal loops bordering nodes of Ranvier and in Schmidt–Lanterman incisures. By immunocytochemistry, connexin32 (Cx32) protein has been demonstrated at paranodal loops and Schmidt–Lanterman incisures, and it is widely assumed that gap junctions are present in these locations, thereby providing a direct radial route for transport of ions and metabolites between cytoplasmic myelin layers. This study used freeze-fracture replica immunogold labeling to detect Cx32 in ultrastructurally defined gap junctions in Schmidt–Lanterman incisures, as well as in a novel location, between the outer two layers of internodal myelin, approximately every micrometer along the entire length of myelin, at the zone between compact myelin and noncompact myelin. Thus, these gap junctions link the partially compacted second layer of myelin to the noncompact outer tongue. Although these gap junctions are unusually small (average, 11 connexon channels), their relative abundance and regular distribution along the zone that is structurally intermediate between compact and noncompact myelin demonstrates the existence of multiple sites for unidirectional or bidirectional transport of water, ions, and small molecules between these two distinct cytoplasmic compartments, possibly to regulate or facilitate myelin compaction or to maintain the transition zone between noncompact and compact myelin.

The extent and strength of electrical coupling between inferior olivary neurons is heterogeneous

Gap junctions constitute the only form of synaptic communication between neurons in the inferior olive (IO), which gives rise to the climbing fibers innervating the cerebellar cortex. Although its exact functional role remains undetermined, electrical coupling was shown to be necessary for the transient formation of functional compartments of IO neurons and to underlie the precise timing of climbing fibers required for cerebellar learning. So far, most functional considerations assume the existence of a network of permanently and homogeneously coupled IO neurons. Contrasting this notion, our results indicate that coupling within the IO is highly variable. By combining tracer-coupling analysis and paired electrophysiological recordings, we found that individual IO neurons could be coupled to a highly variable number of neighboring neurons. Furthermore, a given neuron could be coupled at remarkably different strengths with each of its partners. Freeze-fracture analysis of IO glomeruli revealed the close proximity of glutamatergic postsynaptic densities to connexin 36-containing gap junctions, at distances comparable to separations between chemical transmitting domains and gap junctions in goldfish mixed contacts, where electrical coupling was shown to be modulated by the activity of glutamatergic synapses. On the basis of structural and molecular similarities with goldfish mixed synapses, we speculate that, rather than being hardwired, variations in coupling could result from glomerulus-specific long-term modulation of gap junctions. This striking heterogeneity of coupling might act to finely influence the synchronization of IO neurons, adding an unexpected degree of complexity to olivary networks.

Trafficking of gap junction channels at a vertebrate electrical synapse in vivo

Trafficking and turnover of transmitter receptors required to maintain and modify the strength of chemical synapses have been characterized extensively. In contrast, little is known regarding trafficking of gap junction components at electrical synapses. By combining ultrastructural and in vivo physiological analysis at identified mixed (electrical and chemical) synapses on the goldfish Mauthner cell, we show here that gap junction hemichannels are added at the edges of GJ plaques where they dock with hemichannels in the apposed membrane to form cell–cell channels and, simultaneously, that intact junctional regions are removed from centers of these plaques into either presynaptic axon or postsynaptic dendrite. Moreover, electrical coupling is readily modified by intradendritic application of peptides that interfere with endocytosis or exocytosis, suggesting that the strength of electrical synapses at these terminals is sustained, at least in part, by fast (in minutes) turnover of gap junction channels. A peptide corresponding to a region of the carboxy terminus that is conserved in Cx36 and its two teleost homologs appears to interfere with formation of new gap junction channels, presumably by reducing insertion of hemichannels on the dendritic side. Thus, our data indicate that electrical synapses are dynamic structures and that their channels are turned over actively, suggesting that regulated trafficking of connexons may contribute to the modification of gap junctional conductance.

Gap junction assembly: roles for the formation plaque and regulation by the C-terminus of connexin43

Gap junction (GJ) “formation plaques” are distinct membrane domains with GJ precursors; they assemble by means of a series of defined steps. The C-terminus of Cx43 is required for normal progression of assembly, normal aggregation of 10-nm particles into small GJs, and negative regulation of assembly involving protein kinase C.