Aquaporin (AQP) water channels are abundant in the brain and spinal cord, where AQP1 and AQP4 are believed to play major roles in water metabolism and osmoregulation. Immunocytochemical analysis of the brain recently revealed that AQP4 has a highly polarized distribution, with marked expression in astrocyte end-feet that surround capillaries and form the glia limitans; however, the structural organization of AQP4 has remained unknown. In freezefracture replicas, astrocyte end-feet contain abundant square arrays of intramembrane particles that parallel the distribution of AQP4. To determine whether astrocyte and ependymocyte square arrays contain AQP4, we employed immunogold labeling of SDS-washed freeze-fracture replicas and stereoscopic confirmation of tissue binding. Antibodies to AQP4 directly labeled Ϸ33% of square arrays in astrocyte and ependymocyte plasma membranes in rat brain and spinal cord. Overall, 84% of labels were present beneath square arrays; 11% were beneath particle clusters that resembled square arrays that had been altered during fixation or cleaving; and 5% were beneath the much larger areas of glial plasma membrane that were devoid of square arrays. Based on this evidence that AQP4 is concentrated in glial square arrays, freeze-fracture methods may now provide biophysical insights regarding neuropathological states in which abnormal f luid shifts are accompanied by alterations in the aggregation state or the molecular architecture of square arrays.

The transmembrane connexin proteins of gap junctions link extracellularly to form channels for cell-to-cell exchange of ions and small molecules. Two primary hypotheses of gap junction coupling in the CNS are the following: (1) generalized coupling occurs between neurons and glia, with some connexins expressed in both neurons and glia, and (2) intercellular junctional coupling is restricted to specific coupling partners, with different connexins expressed in each cell type. There is consensus that gap junctions link neurons to neurons and astrocytes to oligodendrocytes, ependymocytes, and other astrocytes. However, unresolved are the existence and degree to which gap junctions occur between oligodendrocytes, between oligodendrocytes and neurons, and between astrocytes and neurons. Using light microscopic immunocytochemistry and freezefracture replica immunogold labeling of adult rat CNS, we investigated whether four of the best-characterized CNS connexins are each present in one or more cell types, whether oligodendrocytes also share gap junctions with other oligodendrocytes or with neurons, and whether astrocytes share gap junctions with neurons. Connexin32 (Cx32) was found only in gap junctions of oligodendrocyte plasma membranes, Cx30 and Cx43 were found only in astrocyte membranes, and Cx36 was only in neurons. Oligodendrocytes shared intercellular gap junctions only with astrocytes, with each oligodendrocyte isolated from other oligodendrocytes except via astrocyte intermediaries. Finally, neurons shared gap junctions only with other neurons and not with glial cells. Thus, the different cell types of the CNS express different connexins, which define separate pathways for neuronal versus glial gap junctional communication.Key words: astrocyte; connexin; connexon; gap junction; neuron; oligodendrocyte Astrocytes, ependymocytes, and oligodendrocytes, the macroglial cells of the adult CNS, are richly invested with gap junctions. Astrocytes, in particular, share gap junctions with all three macroglia, thereby creating a functional panglial syncytium (Mugnaini, 1986;Rash et al., 1997). In contrast, gap junctions involving neurons were reported to be rare (Brightman and Reese, 1969;Sotelo and Korn, 1978), with glial gap junctions greatly outnumbering neuronal gap junctions and neuron-to-glial junctions not detected (Wolff et al., 1998;Rash et al., 2000). The initial "restricted coupling partner" hypothesis that oligodendrocytes share intercellular gap junctions only with astrocytes and that neurons share gap junctions only with neurons (Massa and Mugnaini, 1982;Mugnaini, 1986;Rash et al., 1997) was supported by immunocytochemical data showing that neurons and glia express different connexins Condorelli et al., 1998;Nagy et al., 1999;Nagy and Rash, 2000;Rash et al., 2000). A quite different "shared-connexins/mixed-coupling" hypothesis, which arose from in situ hybridization, imaging of calcium waves, electrical and dye coupling, and immunocytochemistry, suggests that neurons and glia coexpress connexin32 (Cx32) and...

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 A quaporins are specialized water transport channels in plasma membranes of water-permeable tissues (1). Aquaporins 1 and 4 (AQP1 and AQP4) are most important to fluid movements in mammalian brain. AQP4 exists as two isoforms, differing at their N termini, because of translation initiation at the first methionine (M1, 323 aa) or the second methionine (M23, 301 aa) (2, 3). Both isoforms are present in brain, but M23 is at least 3-fold more abundant (4, 5). Endogenous AQP4 is a tetramer usually containing M1 and M23 subunits. The water permeabilities of M1 and M23 are similar, and functional differences are not known (3, 4).Fluid movements are precisely orchestrated within the rigid cranium to prevent physical damage from swelling or shrinkage. Interfaces between brain parenchyma and cerebrospinal fluid occur around the ventricles, surrounding blood vessels, and at the brain surface. AQP1 is expressed in rat choroid plexus, the site of cerebrospinal fluid secretion (6), whereas AQP4 is enriched in rat astrocyte end-feet surrounding brain capillaries (7,8). Astrocyte processes forming the glia limitans at brain surfaces, ependymal cells lining brain ventricles, and Müller cells facing the vitreous body and retinal blood vessels all have abundant AQP4 (9). AQP4 in perivascular membranes of astrocyte end-feet has been implicated in neurological disorders, including acute hyponatremic edema, postischemic injury, and epileptic seizures (10-13).Perivascular membranes of astrocyte end-feet contain numerous strikingly regular arrays of intramembrane particles (IMPs) in freeze-fracture electron micrographs. These IMP arrays have been referred to as square arrays, assemblies, or orthogonally arranged particles (OAPs) (14). In early freeze-fracture images of astrocyte end-feet (15), square arrays were resolved as 6-nm IMP protrusions in P-face images (protoplasmic leaflets) or as smaller pits in E-faces (extraplasmic leaflets). The sizes and shapes of square arrays vary, but the IMPs and pits have un...