Connexin composition in apposed gap junction hemiplaques revealed by matched double-replica freeze-fracture replica immunogold labeling.

Despite the combination of light-microscopic immunocytochemistry, histochemical mRNA detection techniques and protein reporter systems, progress in identifying the protein composition of neuronal versus glial gap junctions, determination of the differential localization of their constituent connexin proteins in two apposing membranes and understanding human neurological diseases caused by connexin mutations has been problematic due to ambiguities introduced in the cellular and subcellular assignment of connexins. Misassignments occurred primarily because membranes and their constituent proteins are below the limit of resolution of light microscopic imaging techniques. Currently, only serial thin-section transmission electron microscopy and freeze-fracture replica immunogold labeling have sufficient resolution to assign connexin proteins to either or both sides of gap junction plaques.

Mixed Electrical-Chemical Synapses in Adult Rat Hippocampus are Primarily Glutamatergic and Coupled by Connexin-36.

Dendrodendritic electrical signaling via gap junctions is now an accepted feature of neuronal communication in mammalian brain, whereas axodendritic and axosomatic gap junctions have rarely been described. We present ultrastructural, immunocytochemical, and dye-coupling evidence for "mixed" (electrical/chemical) synapses on both principal cells and interneurons in adult rat hippocampus. Thin-section electron microscopic images of small gap junction-like appositions were found at mossy fiber (MF) terminals on thorny excrescences of CA3 pyramidal neurons (CA3pyr), apparently forming glutamatergic mixed synapses.

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.

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

Using an established gap junction (GJ) assembly system with experimentally reaggregated cells, we analyzed "formation plaques" (FPs), apparent sites of GJ assembly. Employing freeze-fracture electron microscopy methods combined with filipin labeling of sterols and immunolabeling for connexin43 (Cx43), we demonstrated that FPs constitute distinct membrane "domains" and that their characteristic 10-nm particles contain connexin43, thus representing precursors (i.e.

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.

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.

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 ( approximately 30-70 nm in diameter) were found between pairs of mossy fiber axons ( approximately 100-200 nm in diameter) in the stratum lucidum of the CA3b field of the rat ventral hippocampus, and one axonal gap junction ( approximately 100 connexons) was found on a mossy fiber axon in the CA3c field of the rat dorsal hippocampus.

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.

Ultrastructural localization of connexins (Cx36, Cx43, Cx45), glutamate receptors and aquaporin-4 in rodent olfactory mucosa, olfactory nerve and olfactory bulb.

Odorant/receptor binding and initial olfactory information processing occurs in olfactory receptor neurons (ORNs) within the olfactory epithelium. Subsequent information coding involves high-frequency spike synchronization of paired mitral/tufted cell dendrites within olfactory bulb (OB) glomeruli via positive feedback between glutamate receptors and closely-associated gap junctions. With mRNA for connexins Cx36, Cx43 and Cx45 detected within ORN somata and Cx36 and Cx43 proteins reported in ORN somata and axons, abundant gap junctions were proposed to couple ORNs.

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.

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.