John E. Rash

Connexins and gap junctions of astrocytes and oligodendrocytes in the CNS

This review article summarizes early and recent literature on the structure, distribution and composition of gap junctions between astrocytes and oligodendrocytes, and the differential expression of glial connexins in adult and developing mammalian CNS. In addition to an overview of the topic, discussion is focused on the organization of homologous gap junctional interactions between astrocytes and between oligodendrocytes as well as on heterologous junctional coupling between astrocytes and oligodendrocytes. The homotypic and heterotypic nature of these gap junctions is related to the connexins known to be produced by glial cells in the intact brain and spinal cord. Emphasis is placed on the ultrastructural level of analysis required to attribute gap junction and connexin deployment to particular cell types and subcellular locations. Our aim is to provide a firm basis for consideration of anticipated rapid advances in understanding of structural relationships of gap junctions and connexins within the glial gap junctional syncytium. Conclusions to date suggest that the glial syncytium is more complex than previously appreciated and that glial pathways of junctional communication may not only be determined by the presence of gap junctions, but also by the connexin composition and conductance regulation of junctional channels.

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.

Connexin26 in adult rodent central nervous system: Demonstration at astrocytic gap junctions and colocalization with connexin30 and connexin43

The connexin family of proteins (Cx) that form intercellular gap junctions in vertebrates is well represented in the mammalian central nervous system. Among these, Cx30 and Cx43 are present in gap junctions of astrocytes. Cx32 is expressed by oligodendrocytes and is present in heterologous gap junctions between oligodendrocytes and astrocytes as well as at autologous gap junctions between successive myelin layers. Cx36 mRNA has been identified in neurons, and Cx36 protein has been localized at ultrastructurally defined interneuronal gap junctions. Cx26 is also expressed in the CNS, primarily in the leptomeningeal linings, but is also reported in astrocytes and in neurons of developing brain and spinal cord. To establish further the regional, cellular, and subcellular localization of Cx26 in neural tissue, we investigated this connexin in adult mouse brain and in rat brain and spinal cord using biochemical and immunocytochemical methods. Northern blotting, western blotting, and immunofluorescence studies indicated widespread and heterogeneous Cx26 expression in numerous subcortical areas of both species. By confocal microscopy, Cx26 was colocalized with both Cx30 and Cx43 in leptomeninges as well as along blood vessels in cortical and subcortical structures. It was also localized at the surface of oligodendrocyte cell bodies, where it was coassociated with Cx32. Freeze‐fracture replica immunogold labeling (FRIL) demonstrated Cx26 in most gap junctions between cells of the pia mater by postnatal day 4. By postnatal day 18 and thereafter, Cx26 was present at gap junctions between astrocytes and in the astrocyte side of most gap junctions between astrocytes and oligodendrocytes. In perinatal spinal cord and in five regions of adult brain and spinal cord examined by FRIL, no evidence was obtained for the presence of Cx26 in neuronal gap junctions. In addition to its established localization in leptomeningeal gap junctions, these results identify Cx26 as a third connexin (together with Cx30 and Cx43) within astrocytic gap junctions and suggest a further level of complexity to the heterotypic connexin channel combinations formed at these junctions. J. Comp. Neurol. 441:302–323, 2001.

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.

Coupling of astrocyte connexins Cx26, Cx30, Cx43 to oligodendrocyte Cx29, Cx32, Cx47: Implications from normal and connexin32 knockout mice

Oligodendrocytes in vivo form heterologous gap junctions with astrocytes. These oligodendrocyte/astrocyte (A/O) gap junctions contain multiple connexins (Cx), including Cx26, Cx30, and Cx43 on the astrocyte side, and Cx32, Cx29, and Cx47 on the oligodendrocyte side. We investigated connexin associations at A/O gap junctions on oligodendrocytes in normal and Cx32 knockout (KO) mice. Immunoblotting and immunolabeling by several different antibodies indicated the presence of Cx32 in liver and brain of normal mice, but the absence of Cx32 in liver and brain of Cx32 KO mice, confirming the specificity and efficacy of the antibodies, as well as allowing the demonstration of Cx32 expression by oligodendrocytes. Oligodendrocytes throughout brain were decorated with numerous Cx30‐positive puncta, which also were immunolabeled for both Cx32 and Cx43. In Cx32 KO mice, astrocytic Cx30 association with oligodendrocyte somata was nearly absent, Cx26 was partially reduced, and Cx43 was present in abundance. In normal and Cx32 KO mice, oligodendrocyte Cx29 was sparsely distributed, whereas Cx47‐positive puncta were densely localized on oligodendrocyte somata. These results demonstrate that astrocyte Cx30 and oligodendrocyte Cx47 are widely present at A/O gap junctions. Immunolabeling patterns for these six connexins in Cx32 KO brain have implications for deciphering the organization of heterotypic connexin coupling partners at A/O junctions. The persistence and abundance of Cx43 and Cx47 at these junctions after Cx32 deletion, together with the paucity of Cx29 normally present at these junctions, suggests Cx43/Cx47 coupling at A/O junctions. Reductions in Cx30 and Cx26 after Cx32 deletion suggest that these astrocytic connexins likely form junctions with Cx32 and that their incorporation into A/O gap junctions is dependent on the presence of oligodendrocytic Cx32.

‘NON-SYNAPTIC’ MECHANISMS IN SEIZURES AND EPILEPTOGENESIS

The role of ‘non‐synaptic’ mechanisms (i.e. those mechanisms that are independent of active chemical synpases) in the synchronization of neuronal activity during seizures and their possible contribution to chronic epileptogenesis are summarized. These ‘non‐synaptic’ mechanisms include electrotonic coupling through gap junctions, electrical field effects (i.e. ephaptic transmission), and ionic interactions (e.g. increases in the extracellular concentration of K+). Several lines of evidence indicate that granule cells and pyramidal cells of the hippocampus, and probably other cortical neurons, can generate synchronized electrical activity after active chemical synaptic transmission has been blocked. This synchronized activity is sensitive to alterations in the size of the extracellular space, thus suggesting that electrical field effects and ionic mechanisms contribute to this synchronized activity. Recent studies also indicate that ‘non‐synaptic’ synchronization is quite prominent early in development. Electrophysiological data from hippocampal and neocortical slices have led to a re‐interpretation of the fast prepotentials (i.e. partial spikes) recorded in cortical pyramidal cells, suggesting that they may not be due to dendritic spike generation. Improvement in freeze‐fracture ultrastructural techniques have led to a re‐assessment of previous data on gap junctions in the nervous system and opened new approaches to the quantitative analysis and characterization of gap junctions on glia and neurons. Finally, new methods of dye/tracer coupling have the potential to provide a more rigorous basis for evaluating gap junctions and electrotonic communication between neurons in the mammalian central nervous system. Therefore, recent data continue to suggest that gap junctions and electrotonic coupling play an important role in neural integration, although additional studies using new techniques will be needed to address some of the controversial issues that have arisen over the last several decades.

Neuronal connexin36 association with zonula occludens-1 protein (ZO-1) in mouse brain and interaction with the first PDZ domain of ZO-1

Among the 20 members in the connexin family of gap junction proteins, only connexin36 (Cx36) is firmly established to be expressed in neurons and to form electrical synapses at widely distributed interneuronal gap junctions in mammalian brain. Several connexins have recently been reported to interact with the PDZ domain‐containing protein zonula occludens‐1 (ZO‐1), which was originally considered to be associated only with tight junctions, but has recently been reported to associate with other structures including gap junctions in various cell types. Based on the presence of sequence corresponding to a putative PDZ binding motif in Cx36, we investigated anatomical relationships and molecular association of Cx36 with ZO‐1. By immunofluorescence, punctate Cx36/ZO‐1 colocalization was observed throughout the central nervous system of wild‐type mice, whereas labelling for Cx36 was absent in Cx36 knockout mice, confirming the specificity of the anti‐Cx36 antibodies employed. By freeze‐fracture replica immunogold labelling, Cx36 and ZO‐1 in brain were found colocalized within individual ultrastructurally identified gap junction plaques, although some plaques contained only Cx36 whereas others contained only ZO‐1. Cx36 from mouse brain and Cx36‐transfected HeLa cells was found to coimmunoprecipitate with ZO‐1. Unlike other connexins that bind the second of the three PDZ domains in ZO‐1, glutathione S‐transferase‐PDZ pull‐down and mutational analyses indicated Cx36 interaction with the first PDZ domain of ZO‐1, which required at most the presence of the four c‐terminus amino acids of Cx36. These results demonstrating a Cx36/ZO‐1 association suggest a regulatory and/or scaffolding role of ZO‐1 at gap junctions that form electrical synapses between neurons in mammalian brain.

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.

Direct immunogold labeling of connexins and aquaporin-4 in freeze-fracture replicas of liver, brain, and spinal cord: factors limiting quantitative analysis

Abstract Direct immunogold labeling and histological mapping of membrane proteins is demonstrated in Lexan-stabilized SDS-washed freeze-fracture replicas of complex tissues. Using rat brain and spinal cord as primary model systems and liver as a ”control” tissue to identify preparation and labeling artifacts, we demonstrate the presence of connexin43 in freeze-fractured gap junctions of identified and mapped astrocytes and ependymocytes, and confirm the presence of connexin32 in freeze-fractured gap junctions in liver. In addition, the simultaneous double-labeling of dissimilar proteins (connexin43 and aquaporin-4) is demonstrated in gap junctions and square arrays, respectively, in the plasma membranes of astrocytes and ependymocytes. Finally, double-side shadowing and conventional staining methods are used to reveal the extent of biological material present at the time of labeling and to investigate the dynamics of membrane solubilization, the primary artifacts that occur during labeling, and several factors limiting quantitative analysis.

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.