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...

The subcellular distributions and co-associations of the gap junction-forming proteins connexin 47 and connexin 32 were investigated in oligodendrocytes of adult mouse and rat CNS. By confocal immunofluorescence light microscopy, abundant connexin 47 was co-localized with astrocytic connexin 43 on oligodendrocyte somata, and along myelinated fibers, whereas connexin 32 without connexin 47 was co-localized with contactin-associated protein (caspr) in paranodes. By thin-section transmission electron microscopy, connexin 47 immunolabeling was on the oligodendrocyte side of gap junctions between oligodendrocyte somata and astrocytes. By freeze-fracture replica immunogold labeling, large gap junctions between oligodendrocyte somata and astrocyte processes contained much more connexin 47 than connexin 32. Along surfaces of internodal myelin, connexin 47 was several times as abundant as connexin 32, and in the smallest gap junctions, often occurred without connexin 32. In contrast, connexin 32 was localized without connexin 47 in newly-described autologous gap junctions in Schmidt-Lanterman incisures and between paranodal loops bordering nodes of Ranvier. Thus, connexin 47 in adult rodent CNS is the most abundant connexin in most heterologous oligodendrocyte-to-astrocyte gap junctions, whereas connexin 32 is the predominant if not sole connexin in autologous ("reflexive") oligodendrocyte gap junctions. These results clarify the locations and connexin compositions of heterologous and autologous oligodendrocyte gap junctions, identify autologous gap junctions at paranodes as potential sites for modulating paranodal electrical properties, and reveal connexin 47-containing and connexin 32-containing gap junctions as conduits for long-distance intracellular and intercellular movement of ions and associated osmotic water. The autologous gap junctions may regulate paranodal electrical properties during saltatory conduction. Acting in series and in parallel, autologous and heterologous oligodendrocyte gap junctions provide essential pathways for intra- and intercellular ionic homeostasis.

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 ultrastructuraly-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 astrocytc gap junctions. Oligodendrocytes shared intercellular gap junctions only with astrocytcs, 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 Ieptomenigeal 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.

Auditory afferents terminating as "large myelinated club endings" on goldfish Mauthner cells are identifiable "mixed" (electrical and chemical) synaptic terminals that offer the unique opportunity to correlate physiological properties with biochemical composition and specific ultrastructural features of individual synapses. By combining confocal microscopy and freeze-fracture replica immunogold labeling (FRIL), we demonstrate that gap junctions at these synapses contain connexin35 (Cx35). This connexin is the fish ortholog of the neuron-specific human and mouse connexin36 that is reported to be widely distributed in mammalian brain and to be responsible for electrical coupling between many types of neurons. Similarly, connexin35 was found at gap junctions between neurons in other brain regions, suggesting that connexin35-mediated electrical transmission is common in goldfish brain. Conductance of gap junction channels at large myelinated club endings is known to be dynamically modulated by the activity of their colocalized glutamatergic synapses. We show evidence by confocal microscopy for the presence of the NR1 subunit of the NMDA glutamate receptor subtype, proposed to be a key regulatory element, at these large endings. Furthermore, we also show evidence by FRIL double-immunogold labeling that the NR1 subunit of the NMDA glutamate receptor is present at postsynaptic densities closely associated with gap junction plaques containing Cx35 at mixed synapses across the goldfish hindbrain. Given the widespread distribution of electrical synapses and glutamate receptors, our results suggest that the plastic properties observed at these identifiable junctions may apply to other electrical synapses, including those in mammalian brain.

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.astrocytes ͉ brain edema ͉ ischemia ͉ stroke ͉ water channels S troke is invariably associated with a brain edema that accounts for much of the morbidity and mortality of this condition. The brain edema is often long lasting and therapyresistant and thus poses a major challenge in the clinic. A better understanding is needed of the molecular mechanisms that promote water flux across the brain-blood interface in the build-up phase and resolution phase of cerebral edema.Aquaporin-4 (AQP4) water channels are strongly enriched in the astrocyte plasma membrane domains that ensheathe the cerebral microvessels (1, 2). It was hypothesized (1) that this perivascular pool of AQP4 could become rate-limiting for water flux in pathophysiological conditions, such as in the reperfusion phase after an ischemic insult. This hypothesis was tested in a model that took advantage of the fact that the perivascular AQP4 pool is anchored through the dystrophin complex (comprising the brain dystrophin isoform DP-71 and ␣-syntrophin) (3). Mice with targeted deletion of ␣-syntrophin displayed a dramatic loss of perivascular AQP4 and a concomitant reduction in the extent of postischemic edema (4). These findings [and experiments in mdx mice (5)] support the idea that the perivascular pool of AQP4 facilitates water flux across the brain-blood interface and offer a mechanistic explanation for the reduction in brain edema formation and dissolution observed in AQP4 Ϫ/Ϫ animals (6, 7) The implication of a sp...

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.

Neuronal gap junctions are abundant in both outer and inner plexiform layers of the mammalian retina. In the inner plexiform layer (IPL), ultrastructurally-identified gap junctions were reported primarily in the functionally-defined and anatomically-distinct ON sublamina, with few reported in the OFF sublamina. We used freeze-fracture replica immunogold labeling and confocal microscopy to quantitatively analyze the morphologies and distributions of neuronal gap junctions in the IPL of adult rat and mouse retina. Under "baseline" conditions (photopic illumination/general anesthesia), 649 neuronal gap junctions immunogold-labeled for connexin36 were identified in rat IPL, of which 375 were photomapped to OFF vs. ON sublaminae. In contrast to previous reports, the volume-density of gap junctions was equally abundant in both sublaminae. Five distinctive morphologies of gap junctions were identified: conventional crystalline and non-crystalline "plaques" (71% and 3%), plus unusual "string" (14%), "ribbon" (7%) and "reticular" (2%) forms. Plaque and reticular gap junctions were distributed throughout the IPL. However, string and ribbon gap junctions were restricted to the OFF sublamina, where they represented 48% of gap junctions in that layer. In string and ribbon junctions, curvilinear strands of connexons were dispersed over 5 to 20 times the area of conventional plaques having equal numbers of connexons. To define morphologies of gap junctions under different light-adaptation conditions, we examined an additional 1150 gap junctions from rats and mice prepared after 30 min of photopic, mesopic and scotopic illumination, with and without general anesthesia. Under these conditions, string and ribbon gap junctions remained abundant in the OFF sublamina and absent in the ON sublamina. Abundant gap junctions in the OFF sublamina of these two rodents with rod-dominant retinas revealed previously-undescribed but extensive pathways for inter-neuronal communication; and the wide dispersion of connexons in string and ribbon gap junctions suggests unique structural features of gap junctional coupling in the OFF vs. ON sublamina.

Each day, approximately 0.5-0.9 l of water diffuses through (primarily) aquaporin-1 (AQP1) channels in the human choroid plexus, into the cerebrospinal fluid of the brain ventricles and spinal cord central canal, through the ependymal cell lining, and into the parenchyma of the CNS. Additional water is also derived from metabolism of glucose within the CNS parenchyma. To maintain osmotic homeostasis, an equivalent amount of water exits the CNS parenchyma by diffusion into interstitial capillaries and into the subarachnoid space that surrounds the brain and spinal cord. Most of that efflux is through AQP4 water channels concentrated in astrocyte endfeet that surround capillaries and form the glia limitans. This report extends the ultrastructural and immunocytochemical characterizations of the crystalline aggregates of intramembrane proteins that comprise the AQP4 "square arrays" of astrocyte and ependymocyte plasma membranes. We elaborate on recent demonstrations in Chinese hamster ovary cells of the effects on AQP4 array assembly resulting from separate vs. combined expression of M1 and M23 AQP4, which are two alternatively spliced variants of the AQP4 gene. Using improved shadowing methods, we demonstrate sub-molecular crossbridges that link the constituent intramembrane particles (IMPs) into regular square lattices of AQP4 arrays. We show that the AQP4 core particle is 4.5 nm in diameter, which appears to be too small to accommodate four monomeric proteins in a tetrameric IMP. Several structural models are considered that incorporate freeze-fracture data for submolecular "cross-bridges" linking IMPs into the classical square lattices that characterize, in particular, naturally occurring AQP4. Keywordscross-bridges; furrows; FRIL; plastic deformation; water homeostasis Precise control of water homeostasis is critical in the brain and spinal cord because even minor changes in water metabolism may result in brain edema and rapid development of potentially fatal compressive forces within the rigid encasement of the skull (King and Agre, 1996). Moderate hyponatremia may result in failure of synaptic transmission or in altered neuronal excitability (Kandel et al., 1992;Jefferys, 1995;Nielsen et al., 1997) and is a contributing factor in the pathogenesis of epilepsy (Dudek et al., 1990;Roper et al., 1992), whereas severe hyponatremia contributes to medical complications that arise from brain and spinal cord NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript trauma, brain tumors, neonatal hydrocephalus, acute water intoxication, and drowning (Johnson and Loewy, 1990;King and Agre, 1996;Nielsen et al., 1997).Rates of secretion vs. resorption of cerebrospinal fluid (CSF) is a major controlling factor in osmoregulation of the CNS. The aqueous component of CSF passes primarily through aquaporin-1 (AQP1) in the choroid plexus epithelium , with the remainder arising from metabolism of glucose, from simple transmembrane diffusion through cell membrane lipid bilayers [reviewed by Agre et al. (2...