Heterotypic Gap Junction Research Articles

A Stochastic Model of Voltage-Gating of Connexin-Based Gap Junction Channels Containing Fast and Slow Gates

Connexins (Cxs), a family of membrane proteins, form gap junction (GJ) channels that provide a direct pathway for electrical and metabolic cell-cell interaction. Each hemichannel in the GJ channel contains fast and slow gates sensitive to transjunctional voltage (Vj). The fast gate operates between open and residual states with conductances of γF,o and γF,res, while the slow gate operates between open (γS,o) and fully closed (γS,closed=0) states; all γs rectify but at different degree. We developed a stochastic 16-state model (S16SM), which extends earlier reported S4SM (Paulauskas et al., 2009) and accounts operation of four gates in series, instead of two, to describe the gating properties of homotypic and heterotypic GJ channels.

Heterotypic gap junctions between two mushroom body modulatory neurons are necessary for Drosophila memory formation

Heterotypic gap junctions between two mushroom body modulatory neurons are necessary for Drosophila memory formation

Diversity and properties of connexin gap junction channels

Gap junction channels are composed of two apposing hemichannels (connexons) in the contiguous cells and provide a direct pathway for electrical and metabolic signaling between adjacent cells. The family of connexin genes comprises 20 members in the mouse and 21 genes in the human genome. Connexins are expressed in all tissues except differentiated skeletal muscle, erythrocytes, and mature sperm cells. Various tissues express more than one type of connexins; therefore, homotypic, heterotypic, and heteromeric gap junction channels may form between cells. In this article, we briefly review basic gating and permeability properties of homotypic and heterotypic gap junction channels as well as recent achievements in the research of their regulation by transjunctional voltage, intracellular calcium, pH, and phosphorylation.

Gating Modulation of Connexin45 Gap Junction Channels By Intracellular pH

Intracellular pH (pHi) changes considerably during pathological conditions such as ischemia or epilepsy. Changes of pHi affect the way cells communicate through gap junction (GJ) channels, and therefore disturb normal tissue function. To study pHi-dependent modulation of GJ channels, we used HeLa cells expressing connexin45 (Cx45) or its fusion form with EGFP. The latter along with electrophysiological data allowed us to estimate the proportion of functional channels (NF) within a junctional plaque (JP). We examined how junctional conductance (gj) depends on pHi and how pHi affects voltage-gating properties of Cx45 homotypic and Cx45/Cx43-EGFP heterotypic GJs. Even at pHi= ∼8, where the probability of the channels being fully open approximates 1, gj was maximal but only 5 % of the channels were functional. Changes in pHi from ∼7.2 to ∼8 increased gj ∼1.8-fold in homotypic Cx45 GJs; gj-pHi dependence was sigmoidal with pKa = ∼7. We used a stochastic four-state model of contingent gating to fit experimental gj-Vj dependence, which allowed us to define parameters characterizing voltage-gating sensitivity (V0and A) and NF. We found that alkalization increases gj mainly by increasing V0, i.e., voltage at which open and closed states of hemichannel are at equilibrium. On the other hand, uncoupling by acidification was due to a decrease of both V0 and NF. In both cases, the constant A, characterizing the steepness of gj changes over Vj remained stable. These results agree with data obtained from heterotypic Cx45/Cx43-EGFP GJs in which pKa= ∼6.7, i.e., in between pKas of Cx43-EGFP and Cx45 homotypic GJs. In summary, pHi modulates Vj-gating that largely explains observed pH-dependent changes of cell-cell coupling.

Heterotypic gap junction channels as voltage-sensitive valves for intercellular signaling

Gap junction (GJ) channels assembled from connexin (Cx) proteins provide a structural basis for direct electrical and metabolic cell-cell communication. By combining fluorescence imaging and dual whole-cell voltage clamp methods, we demonstrate that in response to transjunctional voltage (Vj) Cx43/Cx45 heterotypic GJs exhibit both Vj-gating and dye transfer asymmetries. The later is affected by ionophoresis of charged fluorescent dyes and voltage-dependent gating. We demonstrate that small differences in resting (holding) potentials of communicating cells can fully block (at relative negativity on Cx45 side) or enhance (at relative positivity on Cx45 side) dye transfer. Similarly, series of high frequency Vj pulses resembling bursts of action potentials (APs) can fully block or increase the transjunctional flux (Jj) of dye depending on whether pulses are generated in the cell expressing Cx43 or Cx45, respectively. Asymmetry of Jj-Vj dependence is enhanced or reduced when ionophoresis and Vj-gating act synergistically or antagonistically, whereas single channel permeability (Pgamma) remains unaffected. This modulation of intercellular signaling by Vj can play a crucial role in many aspects of intercellular communication in the adult, in embryonic development, and in tissue regeneration.

Gating, permselectivity and pH‐dependent modulation of channels formed by connexin57, a major connexin of horizontal cells in the mouse retina

Mouse connexin57 (Cx57) is expressed most abundantly in horizontal cells of the retina, and forms gap junction (GJ) channels, which constitute a structural basis for electrical and metabolic intercellular communication, and unapposed hemichannels (UHCs) that are involved in an exchange of ions and metabolites between the cytoplasm and extracellular milieu. By combining fluorescence imaging and dual whole-cell voltage clamp methods, we showed that HeLa cells expressing Cx57 and C-terminally fused with enhanced green fluorescent protein (Cx57-EGFP) form junctional plaques (JPs) and that only cell pairs exhibiting at least one JP demonstrate cell-to-cell electrical coupling and transfer of negatively and positively charged dyes with molecular mass up to approximately 400 Da. The permeability of the single Cx57 GJ channel to Alexa fluor-350 is approximately 90-fold smaller than the permeability of Cx43, while its single channel conductance (57 pS) is only 2-fold smaller than Cx43 (110 pS). Gating of Cx57-EGFP/Cx45 heterotypic GJ channels reveal that Cx57 exhibit a negative gating polarity, i.e. channels tend to close at negativity on the cytoplasmic side of Cx57. Alkalization of pH(i) from 7.2 to 7.8 increased gap junctional conductance (g(j)) of approximately 100-fold with pK(a) = 7.41. We show that this g(j) increase was caused by an increase of both the open channel probability and the number of functional channels. Function of Cx57 UHCs was evaluated based on the uptake of fluorescent dyes. We found that under control conditions, Cx57 UHCs are closed and open at [Ca(2+)](o) = approximately 0.3 mm or below, demonstrating that a moderate reduction of [Ca(2+)](o) can facilitate the opening of Cx57 UHCs. This was potentiated with intracellular alkalization. In summary, our data show that the open channel probability of Cx57 GJs can be modulated by pH(i) with very high efficiency in the physiologically relevant range and may explain pH-dependent regulation of cell-cell coupling in horizontal cell in the retina.

Deletion of Astrocyte Connexins 43 and 30 Leads to a Dysmyelinating Phenotype and Hippocampal CA1 Vacuolation

Astrocytes are coupled via gap junctions (GJs) comprising connexin 43 (Cx43) (Gja1) and Cx30 (Gjb6), which facilitate intercellular exchange of ions. Astrocyte connexins also form heterotypic GJs with oligodendrocytic somata and lamellae. Loss of oligodendrocyte gap junctions results in oligodendrocyte and myelin pathology. However, whether loss of astrocyte GJs affects oligodendrocytes and myelin is not known. To address this question, mice with astrocyte-targeted deletion of Cx43 and global loss of Cx30 [double knock-out (dKO)] were studied using Western blotting, immunohistochemistry, electron microscopy, and functional assays. Commencing around postnatal day 23 and persisting into old age, we found widespread pathology of white matter tracts comprising vacuolated oligodendrocytes and intramyelinic edema. In contrast, gray matter pathology was restricted to the CA1 region of the hippocampus, and consisted of edematous astrocytes. No differences were observed in synaptic density or total NeuN(+) cells in the hippocampus, or olig2(+) cells in the corpus callosum. However, in dKO mice, fewer CC1-positive mature oligodendrocytes were detected, and Western blotting indicated reduced myelin basic protein. Pathology was not noted in mice expressing a single allele of either Cx43 or Cx30. When compared with single connexin knock-outs, dKO mice were impaired in sensorimotor (rotarod, balance beam assays) and spatial memory tasks (object recognition assays). We conclude that loss of astrocytic GJs can result in white matter pathology that has functional consequences.

A Stochastic Four-State Model of Contingent Gating of Gap Junction Channels Containing Two “Fast” Gates Sensitive to Transjunctional Voltage

Connexins, a family of membrane proteins, form gap junction (GJ) channels that provide a direct pathway for electrical and metabolic signaling between cells. We developed a stochastic four-state model describing gating properties of homotypic and heterotypic GJ channels each composed of two hemichannels (connexons). GJ channel contain two “fast” gates (one per hemichannel) oriented opposite in respect to applied transjunctional voltage (Vj). The model uses a formal scheme of peace-linear aggregate and accounts for voltage distribution inside the pore of the channel depending on the state, unitary conductances and gating properties of each hemichannel. We assume that each hemichannel can be in the open state with conductance γh,o and in the residual state with conductance γh,res, and that both γh,o and γh,res rectifies. Gates can exhibit the same or different gating polarities. Gating of each hemichannel is determined by the fraction of Vj that falls across the hemichannel, and takes into account contingent gating when gating of one hemichannel depends on the state of apposed hemichannel. At the single-channel level, the model revealed the relationship between unitary conductances of hemichannels and GJ channels and how this relationship is affected by γh,o and γh,res rectification. Simulation of junctions containing up to several thousands of homotypic or heterotypic GJs has been used to reproduce experimentally measured macroscopic junctional current and Vj-dependent gating of GJs formed from different connexin isoforms. Vj-gating was simulated by imitating several frequently used experimental protocols: 1), consecutive Vj steps rising in amplitude, 2), slowly rising Vj ramps, and 3), series of Vj steps of high frequency. The model was used to predict Vj-gating of heterotypic GJs from characteristics of corresponding homotypic channels. The model allowed us to identify the parameters of Vj-gates under which small changes in the difference of holding potentials between cells forming heterotypic junctions effectively modulates cell-to-cell signaling from bidirectional to unidirectional. The proposed model can also be used to simulate gating properties of unapposed hemichannels.

Ablation of Cx47 in transgenic mice leads to the loss of MUPP1, ZONAB and multiple connexins at oligodendrocyte–astrocyte gap junctions

Oligodendrocytes in CNS are linked to astrocytes by heterotypic gap junctions composed of Cx32 and Cx47 in oligodendrocytes and Cx30 and Cx43 in astrocytes. These gap junctions also harbour regulatory proteins, including ZO-1 and ZONAB. Here, we investigated the localization of multi-PDZ domain protein 1 (MUPP1) at these gap junctions and examined accessory proteins and connexins associated with oligodendrocytes in Cx47-knockout mice. In every CNS region tested, punctate immunolabelling for MUPP1 was found on all oligodendrocyte somata in wild-type mice. These MUPP1-positive puncta were colocalized with punctate labelling for oligodendrocytic Cx32 or Cx47, and with astrocytic Cx30 or Cx43 at oligodendrocyte-astrocyte (O/A) gap junctions, but were not found at astrocyte-astrocyte gap junctions. In Cx47-knockout mice, immunolabelling of MUPP1 and ZONAB was absent on oligodendrocytes, whereas some ZO-1-positive puncta remained. In Cx32-knockout mice, MUPP1 and ZONAB persisted at O/A gap junctions. The absence of Cx47 in Cx47-knockout mice was accompanied by a total loss of punctate labelling for Cx30, Cx32 and Cx43 on oligodendrocyte somata, and by a dramatic increase in immunolabelling for Cx32 along myelinated fibers. These results demonstrate MUPP1 at O/A gap junctions and Cx47-dependent targeting of connexins to the plasma membranes of oligodendrocyte somata. Further, it appears that deficits in myelination reported in Cx47-knockout mice may arise not only from a loss of Cx47 but also from the accompanied loss of gap junctions and their regulatory proteins at oligodendrocyte somata, and that loss of Cx47 may be partly compensated for by elevated levels of Cx32 along myelinated fibers.

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. 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.

Characterisation of an intestinal neoplasm modifier locus in Apc Min mice

Intercellular communication is essential for glandular functions and tissue homeostasis. Gap junctions couple cells homotypically and heterotypically and co-ordinate reciprocal responses between the different cell types. Connexins (Cxs) are the main mammalian gap junction proteins, and the distribution of some Cx subtypes in the heterotypic gap junctions is not symmetrical; in the murine mammary gland, Cx26, Cx30 and Cx32 are expressed only in the luminal epithelial cells and Cx43 is expressed only in myoepithelial cells. Expression of all four Cxs peaks during late pregnancy and throughout lactation suggesting essential roles for these proteins in the functional secretory activity of the gland. Transgenic (Tg) mice over-expressing Cx26 driven by keratin 5 promoter had an unexpected mammary phenotype: the mothers were unable to feed their pups to weaning age leading to litter starvation and demise in early to mid-lactation. The mammary gland of K5-Cx26 female mice developed normally and produced normal levels of milk protein, suggesting a defect in delivery rather than milk production. Because the mammary gland of K5-Cx26 mothers contained excessive milk, we hypothesized that the defect may be in an inability to eject the milk. Using ex vivo three-dimensional mammary organoid cultures, we showed that tissues isolated from wild-type FVB females contracted upon treatment with oxytocin, whereas, organoids from Tg mice failed to do so. Unexpectedly, we found that ectopic expression of Cx26 in myoepithelial cells altered the expression of endogenous Cx43 resulting in impaired gap junction communication, demonstrated by defective dye coupling in mammary epithelial cells of Tg mice. Inhibition of gap junction communication or knock-down of Cx43 in organoids from wild-type mice impaired contraction in response to oxytocin, recapitulating the observations from the mammary glands of Tg mice. We conclude that Cx26 acts as a trans-dominant negative for Cx43 function in myoepithelial cells, highlighting the importance of cell type-specific expression of Cxs for optimal contractile function of the mammary myoepithelium.

UNC-1 Regulates Gap Junctions Important to Locomotion in C. elegans

In C. elegans, loss-of-function (lf) mutations of the stomatin-like protein (SLP) UNC-1 and the innexin UNC-9 inhibit locomotion [1, 2] and modulate sensitivity to volatile anesthetics [3, 4]. It was unknown why unc-1(lf) and unc-9(lf) mutants have similar phenotypes. We tested the hypothesis that UNC-1 is a regulator of gap junctions formed by UNC-9. Analyses of junctional currents between body-wall muscle cells showed that electrical coupling was inhibited to a similar degree in unc-1(lf), unc-9(lf), and unc-1(lf);unc-9(lf) double mutants, suggesting that UNC-1 and UNC-9 function together. Expression of Punc-1::DsRED2 and Punc-9::GFP transcriptional fusions suggests that unc-1 and unc-9 are coexpressed in neurons and body-wall muscle cells. Immunohistochemistry showed that UNC-1 and UNC-9 colocalized at intercellular junctions and that unc-1(lf) did not alter UNC-9 expression or subcellular localization. Bimolecular fluorescence complementation (BiFC) assays suggest that UNC-1 and UNC-9 are physically very close at intercellular junctions. Targeted rescue experiments suggest that UNC-9 and UNC-1 function predominantly in neurons to control locomotion. Thus, in addition to the recently reported function of regulating mechanosensitive ion channels [5, 6], SLPs might have a novel function of regulating gap junctions.

Human connexin26 and connexin30 form functional heteromeric and heterotypic channels

Mutations in GJB2 and GJB6, the genes that encode the human gap junction proteins connexin26 (Cx26) and connexin30 (Cx30), respectively, cause hearing loss. Cx26 and Cx30 are both expressed in the cochlea, leading to the potential formation of heteromeric hemichannels and heterotypic gap junction channels. To investigate their interactions, we expressed human Cx26 and Cx30 individually or together in HeLa cells. When they were expressed together, Cx26 and Cx30 appeared to interact directly (by their colocalization in gap junction plaques, by coimmunoprecipitation, and by fluorescence resonance energy transfer). Scrape-loading cells that express either Cx26 or Cx30 demonstrated that Cx26 homotypic channels robustly transferred both cationic and anionic tracers, whereas Cx30 homotypic channels transferred cationic but not anionic tracers. Cells expressing both Cx26 and Cx30 also transferred both cationic and anionic tracers by scrape loading, and the rate of calcein (an anionic tracer) transfer was intermediate between their homotypic counterparts by fluorescence recovery after photobleaching. Fluorescence recovery after photobleaching also showed that Cx26 and Cx30 form functional heterotypic channels, allowing the transfer of calcein, which did not pass the homotypic Cx30 channels. Electrophysiological recordings of cell pairs expressing different combinations of Cx26 and/or Cx30 demonstrated unique gating properties of cell pairs expressing both Cx26 and Cx30. These results indicate that Cx26 and Cx30 form functional heteromeric and heterotypic channels, whose biophysical properties and permeabilities are different from their homotypic counterparts.

Effect of Transjunctional KCl Gradients on the Spermine Inhibition of Connexin40 Gap Junctions

Spermine inhibits rat connexin40 (Cx40) gap junctions. Glutamate residues at positions 9 and 13 and a basic amino acid (HKH) motif at positions 15–17 on the amino terminal domain are essential for this inhibitory activity. Questions remain as to whether spermine occludes the channel within the ion permeation pathway. To examine this question, cis or trans [KCl] was systematically lowered and the equilibrium dissociation constants (Kd) and kinetics of unilateral spermine block on wild-type Cx40 gap junctions were determined. Asymmetric reductions in the trans [KCl] produced noticeable asymmetric shifts in the V1/2 and Gmin values that progressively resembled Gj-Vj relationships observed in heterotypic connexin gap junction combinations. As cis or trans [KCl] was reduced by 25%, 50%, or 75% relative to the spermine-containing side, the transjunctional voltage (Vj)-dependent Kd values increased or decreased, respectively. The spermine on-rates and off-rates, calculated from the junctional current decay and recovery time constants, were similarly affected. Hill coefficients for the spermine dose-response curves were ∼0.58, indicative of negative cooperativity and possible multiple spermine inhibitory sites. The equivalent “electrical distance” (δ) ranged from 0.61 at 25% cis [KCl] to 1.4 at 25% trans [KCl], with a Hill coefficient of 1.0. Symmetrical reductions in [KCl] resulted in intermediate decreases in the spermine Kds, indicative of a minor electrostatic effect and a more significant effect of the transjunctional KCl electrodiffusion potential on the spermine association and dissociation rates. These data are consistent with a single spermine molecule being sufficient to occlude the Cx40 gap junction channel within the KCl permeation pathway.

Permeability of homotypic and heterotypic gap junction channels formed of cardiac connexins mCx30.2, Cx40, Cx43, and Cx45

We examined the permeabilities of homotypic and heterotypic gap junction (GJ) channels formed of rodent connexins (Cx) 30.2, 40, 43, and 45, which are expressed in the heart and other tissues, using fluorescent dyes differing in net charge and molecular mass. Combining fluorescent imaging and electrophysiological recordings in the same cell pairs, we evaluated the single-channel permeability (P(gamma)). All homotypic channels were permeable to the anionic monovalent dye Alexa Fluor-350 (AF(350)), but mCx30.2 channels exhibited a significantly lower P(gamma) than the others. The anionic divalent dye Lucifer yellow (LY) remained permeant in Cx40, Cx43, and Cx45 channels, but transfer through mCx30.2 channels was not detected. Heterotypic channels generally exhibited P(gamma) values that were intermediate to the corresponding homotypic channels. P(gamma) values of mCx30.2/Cx40, mCx30.2/Cx43, or mCx30.2/Cx45 heterotypic channels for AF(350) were similar and approximately twofold higher than P(gamma) values of mCx30.2 homotypic channels. Permeabilities for cationic dyes were assessed only qualitatively because of their binding to nucleic acids. All homotypic and heterotypic channel configurations were permeable to ethidium bromide and 4,6-diamidino-2-phenylindole. Permeability for propidium iodide was limited only for GJ channels that contain at least one mCx30.2 hemichannel. In summary, we have demonstrated that Cx40, Cx43, and Cx45 are permeant to all examined cationic and anionic dyes, whereas mCx30.2 demonstrates permeation restrictions for molecules with molecular mass over approximately 400 Da. The ratio of single-channel conductance to permeability for AF(350) was approximately 40- to 170-fold higher for mCx30.2 than for Cx40, Cx43, and Cx45, suggesting that mCx30.2 GJs are notably more adapted to perform electrical rather than metabolic cell-cell communication.

Gating Properties of Heterotypic Gap Junction Channels Formed of Connexins 40, 43, and 45

Connexins (Cxs) 40, 43, and 45 are expressed in many different tissues, but most abundantly in the heart, blood vessels, and the nervous system. We examined formation and gating properties of heterotypic gap junction (GJ) channels assembled between cells expressing wild-type Cx40, Cx43, or Cx45 and their fusion forms tagged with color variants of green fluorescent protein. We show that these Cxs, with exception of Cxs 40 and 43, are compatible to form functional heterotypic GJ channels. Cx40 and Cx43 hemichannels are unable or effectively impaired in their ability to dock and/or assemble into junctional plaques. When cells expressing Cx45 contacted those expressing Cx40 or Cx43 they readily formed junctional plaques with cell-cell coupling characterized by asymmetric junctional conductance dependence on transjunctional voltage, V j. Cx40/Cx45 heterotypic GJ channels preferentially exhibit V j-dependent gating transitions between open and residual states with a conductance of ∼42 pS; transitions between fully open and closed states with conductance of ∼52 pS in magnitude occur at substantially lower (∼10-fold) frequency. Cx40/Cx45 junctions demonstrate electrical signal transfer asymmetry that can be modulated between unidirectional and bidirectional by small changes in the difference between holding potentials of the coupled cells. Furthermore, both fast and slow gating mechanisms of Cx40 exhibit a negative gating polarity.

Restoration of connexin26 protein level in the cochlea completely rescues hearing in a mouse model of human connexin30-linked deafness

Mutations in genes coding for connexin26 (Cx26) and/or Cx30 are linked to approximately half of all cases of human autosomal nonsyndromic prelingual deafness. Cx26 and Cx30 are the two major Cx isoforms found in the cochlea, and they coassemble to form hybrid (heteromeric and heterotypic) gap junctions (GJs). This molecular arrangement implies that homomeric GJs would remain in the cochlea if one of the coassembly partners were mutated resulting in null expression. We generated mice in which extra copies of the Cx26 gene were transgenically expressed from a modified bacterial artificial chromosome in a Cx30-/- background. In the absence of the Cx30 gene, Cx26 expressed from extra alleles completely restored hearing sensitivity and prevented hair cell death in deaf Cx30-/- mice. The results indicated that hybrid GJs consisting of Cx26 and Cx30 were not essential for normal hearing in mice and suggested that up-regulation of Cx26 or slowing down its protein degradation might be a therapeutic strategy to prevent and treat deafness caused by Cx30 mutations.

Blocking the glial function suppresses subcutaneous formalin-induced nociceptive behavior in the rat

This study examined whether glial cells in the trigeminal nucleus caudalis (Sp5C) were necessary for orofacial nociception and nociceptive processing induced by subcutaneously (s.c.) injection of 5% formalin into left mystacial vibrissae. The immunohistochemical, immunoelectron microscopical methods and behavior assessment were used in this study. Two hours after administration of carbenoxolone (CBX, a gap junction blocker) or fluorocistrate (FCA, a glail metabolic inhibitor) into the cerebellomedullary cistern, the nociceptive behavior and scratching-cumulative time reduced significantly ( P < 0.01). FCA attenuated obviously the expression of Fos/NeuN-immunoreactive (-IR) neurons (mean ± S.E.M. = 29 ± 2.5) and Fos/glial fibrillary acidic protein (GFAP)-IR astrocytes (7.2 ± 2.2) in Sp5C. CBX decreased the number of Fos/NeuN-IR neurons (25 ± 1.7), but did not affect Fos/GFAP-IR astrocytes (16.2 ± 5.4), compared with vehicle-preadministered rats (Fos/NeuN-IR neurons 135 ± 4.2, and Fos/GFAP-IR astrocytes 25.8 ± 4). Immunoelectron microscopy established that Cx32/Cx43 heterotypic gap junctions (HGJs) were present on junction areas between astrocytes and neurons within Sp5C. The number of HGJs increased significantly following formalin s.c. injection. It suggests that the Sp5C astrocytes may play an active regulating role in orofacial nociception via Cx32/Cx43 HGJs between astrocytes and neurons of Sp5C.

Localization of heterotypic gap junctions composed of connexin45 and connexin36 in the rod pathway of the mouse retina

The primary rod pathway in mammals contains gap junctions between AII amacrine cells and ON cone bipolar cells which relay the rod signal into the cone pathway under scotopic conditions. Two gap junctional proteins, connexin36 (Cx36) and connexin45 (Cx45), appear to play a pivotal role in this pathway because lack of either protein leads to an impairment of visual transmission under scotopic conditions. To investigate whether these connexins form heterotypic gap junctions between ON cone bipolar and AII amacrine cells, we used newly developed Cx45 antibodies and studied the cellular and subcellular distribution of this protein in the mouse retina. Specificity of the Cx45 antibodies was determined, among others, by Western blot and immunostaining of mouse heart, where Cx45 is abundantly expressed. In mouse retina, Cx45 immunosignals were detected in both plexiform layers and the ganglion cell layer. Double staining for Cx45 and Cx36 revealed a partial overlap in the punctate patterns in the ON sublamina of the inner plexiform layer of the retina. We quantified the distributions of these two connexins in the ON sublamina, and detected 30% of the Cx45 signals to be co-localized with or in close apposition to Cx36 signals. Combining immunostaining and intracellular dye injection revealed an overlap or tight association of Cx36 and Cx45 signals on the terminals of injected AII amacrine and two types of ON cone bipolar cells. Our results provide direct evidence for heterotypic gap junctions composed of Cx36 and Cx45 between AII amacrine and certain types of ON cone bipolar cells.

The lumbar spinal cord glial cells actively modulate subcutaneous formalin induced hyperalgesia in the rat

We investigated the response and relationship of glial cells and neurons in lumbar spinal cord to hyperalgesia induced by the unilateral subcutaneous formalin injection into the hindpaw of rats. It was demonstrated that Fos/NeuN immunoreactive (-IR) neurons, glial fibrillary acidic protein (GFAP)-IR astrocytes and OX42-IR microglia were distributed in dorsal horn of lumbar spinal cord, predominantly in the superficial layer. In the time-course studies, GFAP-IR astrocytes were firstly detected, OX42-IR microglia were sequentially observed, Fos/NeuN-IR neurons were found slightly late. Immunoelectron microscopy studies established that many heterotypic gap junctions (HGJs), which consisting of Cx43-IR astrocytic process on one side and Cx32-IR dendrite on the other side, were present in superficial layer of dorsal horn. Ninety-one HGJs were found in 100 areas of experimental rats and occupied 91%, while only 39% HGJs were found in control rats. In experimental rats pretreated with intrathecal (i.t.) application of the carbenoxolone (a gap junction blocker) or fluorocitrate (a glial metabolic inhibitor), the paw withdrawal thermal latency was prolonged than those application of the sterile saline (i.t.). It suggests that spinal cord glial cells may play an important role for modulation of hyperalgesia induced by noxious stimuli through HGJs which located between astrocytes and neurons.