Numerous Gap Junctions Research Articles

Gap Junctions are Found between Iris Sphincter Smooth Muscle Cells but not in the Ciliary Muscle of Human and Monkey Eyes

Physiological studies indicate that in primate ciliary muscle there is little or no spread of electrical activity from cell to cell. To clarify to what extent primate intraocular smooth muscles express the structural correlate of electrical coupling, the localization of gap junctions in the ciliary and the iris sphincter muscle of human and monkey ( Macaca fascicularis) eyes was analysed using electronmicroscopy and connexin43 immunocytochemistry. With both methods, numerous gap junctions were identified in the iris sphincter of both species, while no such junctions were observed between individual smooth muscle cells in the ciliary muscle. Both ciliary and iris sphincter smooth muscle cells were connected by numerous adherens-type junctions. These results further underline that primate ciliary muscle is an atypical smooth muscle which shares many common features with striated skeletal muscle.

Human granulosa cells in culture exhibit functional cyclic AMP-regulated gap junctions.

Numerous gap junctions exist between granulosa cells, between cumulus cells and between cumulus cells and the oocyte. They may play a role in the regulation of both follicular development and oocyte status. We used primary cultures of human granulosa cells to study the molecular nature and functionality of these gap junctions. As shown by a cinemicrographic technique, during the first 3 days of culture, cells flattened and extended in several directions by means of cytoplasmic extensions. An ultrastructural study showed the presence of both intercellular and annular gap junctions after 48 h of culture. As revealed by immunodetection analyses, connexin 43 was present. An analysis using a functional procedure, the gap fluorescence recovery after photobleaching (FRAP) method, indicated that: (i) diffusional communication existed among granulosa cells; (ii) the communication was delayed by treatment with 1-heptanol, a well-documented inhibitor of gap junction permeability; and (iii) permeability was up-regulated by incubation with 8-Br-cAMP, an analogue of cyclic AMP. The detection of connexin 43 and functional gap junctions in networks of cytoplasmic extensions indicated junction formation among cells during culture. In conclusion, our results show that human granulosa cells in culture exhibited functional gap junctions. Connexin 43 was present and the permeability of the gap junctions was up-regulated by cyclic AMP, an important modulator of human granulosa cell function.

Ultrastructure of interstitial cells of Cajal in the canine distal esophagus

The ultrastructure of canine distal esophagus was studied focusing on interstitial cells of Cajal (ICC) and their relationships to nerves and muscle. The distal esophagus consisted of two muscle layers composed of intertwining skeletal and smooth muscle bundles. The ICC formed an interconnecting network and were an integral part of these structures. The ICC communicated with one another and with adjacent smooth muscle cells through numerous gap junctions. The morphology of individual ICC resembled that observed in other gut regions. All interstitial cells were densely innervated. The highest density of ICC, just proximal to the lower esophageal sphincter, coincided with the previously reported highest incidence of occurrence of electrical slow wave type action potentials. Examination of a large number of structural associations of ICC led us to conclude that in the distal esophagus, two networks of ICC and nerves exist, one associated with the inner muscle layer, another associated with the outer muscle layer. These networks are not sheet-like structures, such as the network of ICC in the myenteric plexus or deep muscular plexus of the small intestine, but are three dimensional and are interspersed throughout both muscle layers. The networks do not extend into Auerbach's plexus. The main branches of the networks run along the long axis of the esophagus and seem ideally suited to facilitate communication in this direction. These observations suggest that esophageal interstitial cells are structurally organized in such a manner that they may play a role in pacemaking and neural control of esophageal motility.

Characterization of gap junctions between pairs of Leydig cells from mouse testis.

Leydig cells are coupled in vivo by numerous gap junctions. In vivo and in vitro cells were immunolabeled by connexin 43 (Cx43) but not by Cx26 or Cx32 antibodies; immunoblotting confirmed specificity of Cx43 labeling. Pairs of Leydig cells dissociated from mouse testis were studied by dual whole cell voltage clamp, and a high incidence of dye (n = 20) and electrical coupling (n = 60; > 90%) was found. Coupling coefficients were near 1 and junctional conductance (gj) averaged 7.2 +/- 1.2 nS (SE, n = 40). Large transjunctional voltage (Vj) decreased gj; currents decayed exponentially with time constants of seconds that decreased at greater Vj. The residual conductance at large Vj was at least approximately 40% of the initial conductance. Exposure of cell pairs to saline solutions saturated with CO2 (n = 15) or containing 2 mM halothane (n = 15) or 3.5 mM heptanol (n = 15) rapidly and reversibly reduced gj. In eight cell pairs, gating of single junctional channels was observed during halothane-induced reduction in gj. Most gating events at Vj < 40 mV were fit by a Gaussian distribution with a mean of approximately 100 pS. With Vj > 40 mV, smaller transitions of approximately 30 pS were also recorded, and the frequency and duration of the approximately 100-pS transitions decreased. Also, approximately 70-pS transitions between 30- and 100-pS conductances were observed in the absence of 70-pS transitions to or from the baseline, indicating that the 30-pS conductance was a substate induced by large Vj.

Ischemia-induced cellular redistribution of the astrocytic gap junctional protein connexin43 in rat brain

The distribution and levels of the astrocytic gap junction protein, connexin43 (Cx43) was analyzed in various regions of brain as a function of time after neuronal loss and consequent reactive gliosis induced by bilateral carotid occlusion in rats. In the striatum 2 days after induction of ischemia, immunostaining intensity for Cx43 increased in animals exhibiting mild to moderate striatal damage, whereas areas of reduced staining surrounded by elevated levels of Cx43 immunoreactivity were observed in animals with severe ischemic damage. Immunolabelling of glial cell bodies was evident in ischemic, but not normal, striatum. Similar, though less dramatic, changes were seen at 7 days post-ischemia. Compared with the fine punctate pattern of Cx43 staining seen in normal striatum, ischemic striatal areas contained large aggregates of punctate profiles. In the hippocampus, increased immunostaining was seen at 2 and 7 days post-ischemia and, unlike normal hippocampus, neurons in the CA3 pyramidal cell layer were surrounded by a network of Cx43-immunoreactive puncta at the latter survival time. Immuno-EM analysis of ischemic tissue revealed numerous immunolabelled gap junctions among astrocytic processes in the vicinity of degenerating neurons and elevated levels of intracellular Cx43 immunoreactivity in astrocytic processes and cell bodies. No differences in protein levels or phosphorylation states of Cx43 were detected in either hippocampus or striatum by Western blot analyses of ischemic and control tissue. These results suggest that astrocytes respond to an ischemic insult by reorganizing their gap junctions, that the qualitative nature of their response is dependent on the severity of neuronal damage or loss, and that a pool of Cx43 normally undetectable by immunohistochemistry may contribute to the ischemia-induced elevations of immunolabelling for this protein.

A Correlated Study of Metabolic Cell Communication and Gap Junction Distribution in the Adult Frog Lens

By using low molecular weight dye injection and laser scanning confocal microscopy it has been possible to assess the degree of dye communication in several regions (intraepithelial, epithelium-fibre cell, fibre-epithefium and fibre-fibre) of the adult frog lens. These same areas of the lens have also been examined by freeze fracture electron microscopy for the presence and organisation of gap junctions. Epithelial cell lateral membranes have numerous gap junctions which efficiently transmitted dye to their neighbours though none was detectable in the underlying fibre cells. This was correlated with an apparent absence of gap junctions at the epithelium/fibre cell interface. Dye spread between cortical fibre cells was only observed in a subset of fibres in the bow region of the lens. Neither mature cortical fibres nor immature bow fibres appeared to be dye-coupled and we detected no dye passed from fibre cells to adjacent epithelial cells at their anterior ends. This pattern of dye communication was also correlated with the apparent absence of recognisable gap junctions on the lateral membranes of either the recently differentiated bow fibres or the mature cortical fibres. Classical gap junctions were only found on the membranes of fibres between five and ten cells in from the lens bow, i.e. the subset of fibres which were dye-coupled. No gap junctions were found between deeper cortical fibres or nuclear fibres, although they were characterised by a number of square arrays. Though electrically well coupled the adult frog lens may be relatively poorly dye coupled and this could depend on the age and differentiation stage of the cells concerned. The model of a freely communicating lens clearly requires re-examination by correlated physiological and morphological studies.

Immunolocalization of glucose transporter GLUT1 in the rat placental barrier: possible role of GLUT1 and the gap junction in the transport of glucose across the placental barrier

GLUT1 is an isoform of facilitated-diffusion glucose transporters and has been shown to be abundant in cells of blood-tissue barriers. Using antibodies against GLUT1, we investigated the immunohistochemical localization of GLUT1 in the rat placenta. Rat placenta is of the hemotrichorial type. Three cell layers (from the maternal blood side inward) cytotrophoblast and syncytiotrophoblasts I and II, lie between the maternal and fetal bloodstreams. GLUT1 was abundant along the invaginating plasma membrane facing the cytotrophoblast and the syncytiotrophoblast I. Also, the infolded basal plasma membrane of the syncytiotrophoblast II was rich in GLUT1. Apposing plasma membranes of syncytiotrophoblasts I and II, however, had only a small amount of GLUT1. Numerous gap junctions were seen between syncytiotrophoblasts I and II. Taking into account the localization of GLUT1 and the gap junctions, we suggest a possible major transport route of glucose across the placental barrier, as follows: glucose in the maternal blood passes freely through pores of the cytotrophoblast. Glucose is then transported into the cytoplasm of the syncytiotrophoblast I via GLUT1. Glucose enters the syncytiotrophoblast II through the gap junctions. Finally glucose leaves the syncytiotrophoblast II via GLUT1 and enters the fetal blood through pores of the endothelial cells.

Tissue-specific determinants of anisotropic conduction velocity in canine atrial and ventricular myocardium.

Electrical conduction is very rapid and highly anisotropic in atrial fiber bundles, such as the crista terminalis. In contrast to left ventricular myocardium in which the ratio of longitudinal to transverse conduction velocities is approximately 3, propagation velocity in the crista terminalis is approximately 10 times greater in the longitudinal than in the transverse direction. To elucidate potential determinants of these distinct conduction properties, we characterized structural and molecular features of intercellular coupling in the crista terminalis and left ventricular myocardium of the canine heart. Analysis of the number and spatial orientation of myocyte interconnections at gap junctions revealed that a typical left ventricular myocyte was connected to 11.3 +/- 2.2 other myocytes. Approximately equal numbers of connections occurred between ventricular myocytes juxtaposed in side-to-side and end-to-end orientation. In contrast, a typical myocyte of the crista terminalis was connected to only 6.4 +/- 1.7 other cells (P < .05), but nearly 80% of these connections occurred between cells oriented in an end-to-end configuration. In comparison with the ventricular pattern, this spatial distribution of connections would limit intercellular current transfer between laterally apposed cells and thereby enhance anisotropy of conduction velocity in the longitudinal and transverse directions. Ultrastructural analysis showed that crista terminalis myocytes were connected by numerous small gap junctions that occurred in relatively simple, straight intercalated disks. Northern blot analysis showed approximately equivalent amounts of mRNAs encoding the gap junction channel proteins connexin43 and connexin45 but approximately four times more connexin40 mRNA in crista terminalis than in the left ventricle.(ABSTRACT TRUNCATED AT 250 WORDS)

Morphometric analysis of gap junction density in human myometrium at term

In the human myometrium, the formation of gap junctions in the various stages of labor and, in correlation with the concentration of progesterone in the maternal blood, has not been described adequately. The accepted hypothesis that raised concentrations of progesterone in maternal blood in animals prevent the formation of gap junctions was scrutinized in human myometrium. Myometrial tissue was examined by electron microscopy for the presence of gap junctions and analyzed morphometrically in 17 women who had an elective or emergency cesarean operation at the 38th or 39th week of pregnancy. The concentrations of progesterone and estradiol in the maternal and cord blood was measured by means of radioimmunoassay. The frequency and area of gap junctions varied in relation to the presence or absence of labor. In the myometrium of women in labor, gap junctions were most frequent (5.2/1000 microns of cell membrane), their area largest (0.32 microns 2/1000 microns of cell membrane) and their fractional area greatest (0.20%). When the women were not in genuine labor or when contractions were weak, the percentage fractional areas of gap junctions were low (0.020-0.090) and the gap junctions very small. In the pre- and active phases of labor, the concentration of progesterone in maternal blood was significantly higher (p < 0.025 and 0.03 respectively) than in women who were not in labor. Hence, despite high concentrations of progesterone in the maternal and cord blood at term, the myometrium presented numerous gap junctions of relatively large area.

Structural Basis of Glucose Transport in the Placental Barrier: Role of GLUT1 and the Gap Junction

Glucose transport across cellular membranes is mediated by integral membrane proteins (glucose transporters). GLUT1, an isoform of facilitated-diffusion glucose transporters, is abundant in human and rat placentae. In the human placenta, syncytiotrophoblast serves as the structural basis of the placental barrier. GLUT1 is present along the luminal and basal plasma membranes of this cell. GLUT1 seems to play an important role in the transfer of glucose through a syncytiotrophoblast layer in the human placenta. In the rat placenta, two layers of syncytiotrophoblasts (namely syncytiotrophoblasts I and II from the maternal blood side to the fetal side) serve as a barrier. GLUT1 is abundant in these syncytiotrophoblasts. It is concentrated along the plasma membrane of the maternal side in syncytiotrophoblast I and also along the plasma membrane of the fetal side in the adjacent syncytiotrophoblast II. These two syncytiotrophoblast layers are connected by numerous gap junctions. Connexin 26 is present in these gap junctions. GLUT1 in conjunction with connexin 26 in the gap junction seems to serve as the machinery for trans-placental transfer of glucose in the rat placental barrier.

Interstitial cells in deep muscular plexus of canine small intestine may be specialized smooth muscle cells

The fine structures and properties of cells between the inner and outer circular muscle layers in the canine small intestine were studied by transmission electron microscopy (TEM), immunocytochemistry, and scanning electron microscopy (SEM). A nerve plexus (deep muscular plexus) supported by enteroglial cells, fibroblasts around blood vessels, macrophages, and thin and branched cells previously identified as interstitial cells of Cajal was observed. The interstitial cells of the deep muscular plexus (IC-DMP) were rich in mitochondria, dense bodies, and caveolae, and they were closely associated with nerve fibers. The IC-DMP had incomplete basal laminae. These cells also had numerous interconnecting gap junctions, and they also formed gap junctions with the surrounding smooth muscle cells of the outer circular muscle layer. IC-DMP were rich in myofilaments, which were primarily actin thin filaments, but myosin thick filaments, identified with anti-myosin light-chain antibodies, were also apparent. IC-DMP and circular smooth muscle cells both expressed immunoreactivity to anti-smooth muscle actin antisera, but these two types of cells differed in their intermediate filament proteins: IC-DMP featured vimentin immunopositive filaments, and circular smooth muscle cells featured desmin immunoreactivity. SEM showed that IC-DMP had thin and flat cell bodies with numerous branching processes. These cells came into close contact with nerve fibers and circular smooth muscle cells. The findings that IC-DMP cells contained myosin thick filaments and were immunopositive for anti-smooth muscle actin suggest that they may be more properly categorized as a type of smooth muscle cell.

Dye and electric coupling between osteoblast-like cells in culture

Primary cultures of osteoblast-like cells (OB) derived from calvarial fragments of newborn rats and juvenile guinea pigs formed numerous gap junctions between neighboring cells in vitro. Intracellular injection of Lucifer yellow led to a staining of up to 30 adjacent cells. Parallel intracellular recordings showed that amplitudes of stimulated membrane potential changes (4-5 mV) were closely related between coupled cells. The coupling factor, which was derived from the ratio of these amplitudes, ranged between 0.1 and 0.8. The coupling factor (1) was not dependent on the membrane potential or the injected current strength; (2) strong acidosis (pH < 6.6) and hypercapnia (pCO2 > 80 mm Hg) did not affect electric or dye coupling; (3) elevation of intracellular cAMP level was ineffective; (4) rise of the extra- and intracellular Ca2+ concentration did not effect the electric coupling; (5) the anticonvulsant drugs carbamazepine and phenytoin impaired the coupling factor up to 59%. The findings show that cell-cell communication between OB via gap junctions proved stable under various conditions which, in other tissues, were found to reduce the coupling strength of gap junctions.

Characterization of gap junctions between osteoblast-like cells in culture.

The structure of gap junctions in osteoblast-like cells (OBs) and the connexins (cx) that build up these structures were characterized by ultrastructural, immunocytochemical, and molecular techniques. Ultrastructural studies revealed numerous gap junctions which were mostly located on processes of neighboring cells. Immunofluorescence labeling using two different antibodies (specific to mouse live cx26 and cx32 and to a peptide-specific rat heart gap junction protein cx43) gave evidence that in OBs, gap junctions consist mainly of cx43. The presence of cx43 in cultured OB was also confirmed by Western blot analysis. Dye-coupling with Lucifer yellow led to a staining of up to 30 neighboring cells. Parallel intracellular recordings showed that membrane potential amplitude changes (4-5 mV) are typically related to those in the coupled cells. Thus, there is morphological and functional evidence for intercellular communication between OB in culture. OBs in culture express the same connexins as observed in vivo and may serve as a model to investigate electrophysiological events in response to different stimulation signals.

Membrane architecture as a function of lens fibre maturation: A freeze fracture and scanning electron microscopic study in the human lens

The ultrastructure of fibre membranes in human lenses, varying in age from premature to 40 years, was investigated using a strict protocol regarding their localization within the lens. The ultrastructural approaches used were scanning electron microscopy (SEM), and transmission electron microscopy (TEM) of ultrathin sections and freeze-fracture replicas. Irrespective of the age of the lens, superficial fibre membranes are characterized by a high density of intramembrane particles (IMPs) and numerous gap junctions (GJs). In contrast deep cortical fibres, at the SEM-level characterized by grooves and ridges, are largely free of IMPs but still contain numerous GJs. In between these regions a transitional zone was observed. At the SEM-level the transitional fibres are characterized by wrinkled membranes and formation of grooves and ridges. In freeze-fracture replicas the presence of numerous square arrays (SAs) associated with GJs is most remarkable. It is concluded that at all ages studied, the maturation and compaction of lens fibres results in a transformation of membrane architecture leading to clear-cut ultrastructural differences between superficial and deep cortical membranes. It is argued that this ultrastructural heterogeneity parallels the gradients observed biochemically for intrinsic membrane proteins and cholesterol:phospholipid ratios. The observations confirm the electrophysiological view that superficial membranes have an ‘average’ permeability and that deep cortical membranes are ‘degenerate’ or ‘non-leaky’.

Cytology and organization of reactive astroglia in human cerebellar cortex with severe loss of granule cells: A study on the ataxic form of creutzfeldt-jakob disease

In order to investigate the cellular basis of human astrogliosis, we have selected the cerebellar cortex because it provides a relatively simple and geometrical organization of both neuronal and glial populations. A pathological system with severe and progressive loss of granule cells was studied: the ataxic form of Creutzfeldt-Jakob disease, where the tissue geometry is minimally disturbed. The quantitative study revealed a drastic reduction in the numerical density of granule cells in the Creutzfeldt-Jakob disease cerebellum, and a significant increase in the numerical density of astrocytes. Karyometric analysis showed that the nuclear area was significantly greater in reactive astroglial cells than in normal astroglia. Glial fibrillary acidic protein immunocytochemistry revealed astroglial hypertrophy, but the geometry and spatial domains of astroglial subtypes were strictly preserved. Vimentin expression was detected in Bergmann glia and in certain astrocytes of the granular layer. Ultrastructural analysis showed that reactive astroglia had large nuclei, with expanded interchromatinic regions which contained clusters of interchromatin granules and nuclear bodies, and prominent reticulate nucleoli. In the cytoplasm, hypertrophied bundles of intermediate filaments were observed, some of them associated with the nuclear envelope. Numerous adhering and gap junctions were also found among reactive astroglial cells. Perivascular glial processes showed a terminal web of intermediate filaments and a conspicuous plasmalemmal undercoat. Interendothelial tight junctions were preserved. Our results suggest that the severe loss of granule cells induces a highly ordered astroglial response which tends to preserve the geometry of the astroglial scaffold, the domains of each astroglial subtype, the neuronal microenvironmental conditions and the efficiency of the blood-brain barrier, in order to promote neuron survival.

Ultrastructural observations of typical gap junctions in human foetal lens nucleus

The ultrastructure of gap junctions throughout the human foetal lens was observed. By freeze-fracture analysis, we observed numerous gap junctions in both lens cortex and lens nucleus. Comparison between lens cortex and lens nucleus showed that the gap junctions of lens nucleus are characterized by extreme mosaics of closely apposed P- and E-faces in junctional areas, though no significant difference in the area of gap junctions was observed between lens cortex and lens nucleus. In addition, some morphological variations, such as the smooth domains without particles or pits in junctional areas and the reticulated figures of gap junctions, were observed only in the lens nucleus. We also observed by thin-section electron microscopy that cell membranes of human foetal lens nucleus, as observed in the lens cortex, are mainly composed of continuous lipid bilayer and junctional structures. We concluded that characteristic morphology of lens gap junctions, as observed in the cortex of human foetal lens, is mostly preserved in the human foetal lens nucleus, although some depth-dependent alterations were also observed.

The Advantages of Cryotechniques: Application To Bioluminescent Cells

The use of fast-freeze fixation (FFF) followed by freeze-substitution (FS) brings substantial advantages which are due to the extreme rapidity of this fixation compared to the conventional one. The initial step, FFF, physically immobilizes most molecules and therefore arrests the biological reactions in a matter of milliseconds. The second step, FS, slowly removes the water content still in solid state and, at the same time, chemically fixes the other cell components in absence of external water. This procedure results in an excellent preservation of the ultrastructure, avoids osmotic artifacts,maintains in situ most soluble substances and keeps up a number of cell activities including antigenicities. Another point of interest is that the rapidity of the initial immobilization enables the capture of unstable structures which, otherwise, would slip towards a more stable state. When combined with electrophysiology, this technique arrests the ultrastructural modifications at a well defined state, allowing a precise timing of the events.We studied the epithelium of the elytra of the scale-worm, Harmothoe lunulata which has excitable, conductible and bioluminescent properties. The intracellular sites of the light emission are paracrystals of endoplasmic reticulum (PER), named photosomes (Fig.1). They are able to flash only when they are coupled with plasma membrane infoldings by dyadic or triadic junctions (Fig.2) basically similar to those of the striated muscle fibers. We have studied them before, during and after stimulation. FFF-FS showed that these complexes are labile structures able to diffentiate and dedifferentiate within milliseconds. Moreover, a transient network of endoplasmic reticulum was captured which we have named intermediate endoplasmic reticulum (IER) surrounding the PER (Fig.1). Numerous gap junctions are found in the membranous infoldings of the junctional complexes (Fig.3). When cryofractured, they cleave unusually (Fig.4-5). It is tempting to suggest that they play an important role in the conduction of the excitation.

Permeability of the Pineal Gland of the Viscacha (Lagostomus maximus maximus) to the Lanthanum Hydroxide

Lanthanum hydroxide together with a fixative solution was injected into the third ventricle of the viscacha. The tracer penetrated freely into the pineal gland demonstrating an extensive network of intercommunicating intercellular spaces. Numerous gap junctions were observed indicating that they are the predominant interparenchymal contacts. The capillaries were characterized by non-fenestrated endothelial cells lining a moderate-sized perivascular space. Precipitates of lanthanum hydroxide were found in the basal membranes of the perivascular and interendothelial spaces, but not in the vascular lumina. The tracer stopped at tight junctions; these constitute a barrier between the pinealocytes and the blood compartment. Active micropinocytosis observed in the capillary walls suggest a transendothelial transport.

Direct connections between the R7/8 and R1-6 photoreceptor subsystems in the dipteran visual system.

Musca and related flies have three main photoreceptor subsystems. The R1-6 group has short axons that terminate in the cartridges of the first optic neuropile, the lamina. The cartridges are bypassed by the longer axons of R7 and R8, which run together to terminate at different levels in the underlying medulla neuropile. The present account describes a shallow, previously unidentified zone in the lamina within which R7/8 make glancing contact with R1-6. At the distal border of the cartridge over no more than 3-4 microns depth, the tangentially directed short axon of R6 squeezes between the pair from R7 and R8, forming quite large areas of mutual contact (approximately 7 microns2). Less frequently, R1 is contacted. At least some of these sites contain smaller membrane specialisations indistinguishable from the more numerous gap junctions found more proximally that interconnect the terminals of R1-6. The R7/8 junctions with R6 are of comparable size (0.15 micron 2) and likewise possess symmetrical membrane densities. They provide proposed pathways for direct electrical interaction to account for observed electrical input from R7/8 to the R1-6 subsystem. In two cases R7/8 was possibly postsynaptic to R1-6 at a multiple-contact synapse, but even if functional, these sites were so rare that they are unlikely to have much operational significance.

Correlated electrophysiology and morphology of the ependyma in rat hypothalamus

The ependyma lines the ventricular system of the vertebrate brain and spinal cord. Although its embryology and morphology have been studied extensively, little is known of its physiological properties, particularly in mammals. Tanycytes are modified ependymal cells that are found predominantly lining the floor of the third ventricle, overlying the median eminence. Their processes accompany and enwrap neuroendocrine axons that course from hypothalamic nuclei to terminals in the median eminence, but the significance of this interaction is not yet understood. Intracellular recording and injection techniques were used to study ependymal cells and tanycytes of the rat in the hypothalamic slice preparation after differentiating their respective regions morphologically. With extracellular [K+] = 6.24 mM, the mean membrane potential (+/- SD) for common ependyma was -79.9 +/- 1.40 mV and for tanycytes, -79.5 +/- 1.77 mV. Input resistances (Rin) were very low (much less than 1 M omega). Single-cell injection of Lucifer yellow revealed dye coupling among 2-70 ependymal cells and 5-48 tanycytes. In both freeze-fractured replicas and thin sections, large numbers of gap junctions were found between adjacent ependymal cells and between adjacent tanycytes. The observations of numerous gap junctions, extensive dye coupling and low input resistance demonstrated that both populations are strongly coupled networks. Perhaps for this reason, attempts to uncouple these cells using sodium propionate or CO2 were unsuccessful. Electrical stimulation of the arcuate nucleus did not elicit any detectable synaptic response in impaled tanycytes, so that the functional significance of synaptoid contacts between neuroendocrine neurons and the postsynaptic tanycytes is not yet apparent. Ependymal cells and tanycytes demonstrated a near-Nernstian response to changes in extracellular [K+] between 3 and 20 mM. This finding, as well as their high negative resting potential, low Rin, extensive coupling and absence of spontaneous electrical excitability demonstrate that ependymal cells possess numerous glial characteristics and may therefore have similar functions. In the hypothalamus, ependyma probably take up K+ released from adjacent endocrine neurons and shunt it to the ventricular space.