Defective trafficking and cell death is characteristic of skin disease-associated connexin 31 mutations

Distinct germline mutations in the gene (GJB3) encoding connexin 31 (Cx31) underlie the skin disease erythrokeratoderma variabilis (EKV) or sensorineural hearing loss with/without peripheral neuropathy. Here we describe a number of functional analyses to investigate the effect of these different disease-associated Cx31 mutants on connexon trafficking and intercellular communication. Immunostaining of a biopsy taken from an EKV patient harbouring the R42P mutation revealed sparse epidermal staining of Cx31, and, when present, it had a perinuclear localization. Transfection and microinjection studies in both keratinocytes and fibroblast cell lines also demonstrated that R42P and four other EKV-associated mutant Cx31 proteins displayed defective trafficking to the plasma membrane. The deafness/neuropathy only mutant 66delD had primarily a cytoplasmic localization, but some protein was visualized at the plasma membrane in a few transfected cells. Both 66delD- and R32W-Cx31/EGFP proteins had significantly impaired dye transfer rates compared to wild-type Cx31/EGFP protein. A striking characteristic feature observed with the dominant skin disease Cx31 mutations was a high incidence of cell death. This was not observed with wild-type, R32W 66delD Cx31 proteins. In conclusion, we have identified some key cellular phenotypic differences with respect to disease-associated Cx31 mutations.     

The connexin31 F137L mutant mouse as a model for the human skin disease erythrokeratodermia variabilis (EKV)

Erythrokeratodermia variabilis (EKV) is a rare autosomal dominant human genodermatosis. Its clinical appearance varies from transient, fast moving erythemas to persistent brown hyperkeratoses. So far, several mutations in the Cx31 or Cx30.3 gene have been reported to cause EKV in humans. We have generated a conditional mouse mutant that carries the human F137L mutation in the Cx31 gene which was described to act in a transdominant negative manner. The phenylalanine residue at position 137 is highly conserved in several human and mouse connexin genes. Mouse embryonic stem (ES) cells expressing one allele of the Cx31F137L mutation were stable but showed approximately 30% decreased transfer of neurobiotin. This is probably due to dominant negative effects of the Cx31F137L protein on wild type Cx31 and Cx43 protein expressed in ES cells. Surprisingly, the healing process of tail incision wounds in Cx31(+/F137L) mice was shortened by 1 day, i.e. very similar as previously reported for mice with decreased expression of Cx43 in the epidermis. This suggests again that Cx31 and Cx43 proteins functionally interact, possibly by forming heteromeric channels in the epidermis. Heterozygous Cx31(+/F137L) mice are viable and fertile, in contrast to homozygous Cx31(F137L/F137L) mice that die around ED 7.5. In Cx31(+/F137L) mice, the epidermal expression pattern and level of Cx26, Cx30, Cx30.3 and Cx43 proteins were not altered compared with wild-type mice. No erythemas were detected in young C31(+/F137L) mice before 2 weeks of age. In contrast to human EKV patients, hyperproliferation of the stratum germinativum was found in only 5% of the analyzed skin area.     

A mutation in GJB3 is associated with recessive erythrokeratodermia variabilis (EKV) and leads to defective trafficking of the connexin 31 protein

Erythrokeratodermia variabilis (EKV) is a skin disorder characterized by variable (transient) erythemas and fixed keratosis. The disorder maps to chromosome 1p34-35, a location that contains the GJB3 gene encoding the gap junction protein connexin 31. Until now, only heterozygote mutations in the form of dominant inheritance have been described in this gene associated with EKV. We report here a homozygote mutation in the connexin 31 gene, found in a family that shows recessive inheritance of the disorder, thus providing the first molecular support for a recessive variant of EKV. The entire GJB3 coding sequence was scanned for mutations by sequencing. We detected a T-->C transition at position 101 of the coding sequence, which replaces a leucine with a proline at residue 34 of the protein (L34P). Evolutionary analysis shows that this mutation is located at a highly conserved region of connexin in the first putative transmembrane helix (TMH). In transfected keratinocytes, L34P connexin 31 had a cytoplasmic distribution, suggesting that the mutant form of this protein will not form normal gap junctions between adjacent cells. The change of leucine to proline is likely to alter the structure of the first TMH of connexin by inducing a kink, thus influencing connexon structure and function.     

Chromosomal assignments of mouse connexin genes,coding for gap junctional proteins,by somatic cell hybridization

The connexin genes Cx31 and Cx45 coding for proteins of gap junctional subunits have been assigned to mouse chromosomes 4 and 11 by Southern blot hybridization of specific gene probes to DNA from mouse × Chinese hamster somatic cell hybrids. In addition, our results confirm the recent assignment of mouse connexin genes Cx26, Cx32, Cx37, Cx40, Cx43, and Cx46 to mouse chromosomes 14, X, 4, 3, 10, and 14, respectively, by analysis of interspecific backcrosses and by somatic cell hybridization. Our assignment of the Cx31 gene to mouse chromosome 4 locates the fourth connexin gene on this mouse chromosome to which the genes for Cx31.1, Cx37, and Cx30.3 have previously been assigned. Interestingly three of them (coding for Cx31, Cx31.1, and Cx30.3) are preferentially expressed in skin. Possibly some of the connexin genes clustered on mouse chromosome 4 may be regulated coordinately.     

Tissue-specific effects of wild-type and mutant connexin 31: a role in neurite outgrowth

Channels formed by connexins (Cx), the major protein subunits of gap junctions, allow passage of ions and molecular messengers between cells to provide a mechanism of synchronized cellular response. Twenty human Cx isoforms have been identified and mutations in the gene GJB3 encoding the 31 kDa isoform, Cx31, can cause dominant or recessive skin disease, dominant or recessive deafness or dominant neuropathy with deafness. Cx31 is expressed in differentiating keratinocytes in skin. Here, we also demonstrate endogenous Cx31 expression in human neuronal cell lines, particularly in differentiated neurones. Exogenous Cx31 expression induced neurite outgrowth in human neuronal cell lines, but not differentiation in primary human keratinocytes. Though neither the neuropathy and hearing loss mutation (66delD)Cx31 nor the skin disease associated mutation (R42P)Cx31 is able to traffic to the plasma membrane, the R42P mutant induced neurite outgrowth to a level equal to wild-type Cx31. In contrast, there was significantly reduced neurite outgrowth after (66delD)Cx31 expression. In addition to indicating a potential disease mechanism for the neuropathy/deafness mutation, this work demonstrates a tissue-specific function for Cx31.     

High-resolution proteomic mapping in the vertebrate central nervous system: close proximity of connexin35 to NMDA glutamate receptor clusters and co-localization of connexin36 with immunoreactivity for zonula occludens protein-1 (ZO-1)

Combined confocal microscopy and freeze-fracture replica immunogold labeling (FRIL) were used to examine the connexin identity at electrical synapses in goldfish brain and rat retina, and to test for "co-localization" vs. "close proximity" of connexins to other functionally interacting proteins in synapses of goldfish and mouse brain and rat retina. In goldfish brain, confocal microscopy revealed immunofluorescence for connexin35 (Cx35) and NMDA-R1 (NR1) glutamate receptor protein in Mauthner Cell/Club Ending synapses. By FRIL double labeling, NR1 glutamate receptors were found in clusters of intramembrane particles in the postsynaptic membrane extraplasmic leaflets, and these distinctive postsynaptic densities were in close proximity (0.1-0.3 microm) to neuronal gap junctions labeled for Cx35, which is the fish ortholog of connexin36 (Cx36) found at neuronal gap junctions in mammals. Immunogold labeling for Cx36 in adult rat retina revealed abundant gap junctions, including several previously unrecognized morphological types. As in goldfish hindbrain, immunogold double labeling revealed NR1-containing postsynaptic densities localized near Cx36-labeled gap junction in rat inferior olive. Confocal immunofluorescence microscopy revealed widespread co-localization of Cx36 and ZO-1, particularly in the reticular thalamic nucleus and amygdala of mouse brain. By FRIL, ZO-1 immunoreactivity was co-localized with Cx36 at individual gap junction plaques in rat retinal neurons. As cytoplasmic accessory proteins, ZO-1 and possibly related members of the membrane-associated guanylate kinase (MAGUK) family represent scaffolding proteins that may bind to and regulate the activity of many neuronal gap junctions. These data document the power of combining immunofluorescence confocal microscopy with FRIL ultrastructural imaging and immunogold labeling to determine the relative proximities of proteins that are involved in short- vs. intermediate-range molecular interactions in the complex membrane appositions at synapses between neurons.     

Spatial relationships of connexin36, connexin57 and zonula occludens-1 in the outer plexiform layer of mouse retina

Horizontal cells form gap junctions with each other in mammalian retina, and lacZ reporter analyses have recently indicated that these cells express the Cx57 gene, which codes for the corresponding gap junctional protein. Using anti-connexin57 antibodies, we detected connexin57 protein in immunoblots of mouse retina, and found punctate immunolabeling of this connexin co-distributed with calbindin-positive horizontal cells in the retinal outer plexiform layer. Double immunofluorescence labeling was conducted to determine the spatial relationships of connexin36, connexin57, the gap junction-associated protein zonula occludens-1 and the photoreceptor ribbon synapse-associated protein bassoon in the outer plexiform layer. Connexin36 was substantially co-localized with zonula occludens-1 in the outer plexiform layer, and both of these proteins were frequently located in close spatial proximity to bassoon-positive ribbon synapses. Connexin57 was often found adjacent to, but not overlapping with, connexin36-positive and zonula occludens-1-positive puncta, and was also located adjacent to bassoon-positive ribbon synapses at rod spherules, and intermingled with such synapses at cone pedicles. These results suggest zonula occludens-1 interaction with connexin36 but not with Cx57 in the outer plexiform layer, and an absence of connexin57/connexin36 heterotypic gap junctional coupling in mouse retina. Further, an arrangement of synaptic contacts within rod spherules is suggested whereby gap junctions between horizontal cell terminals containing connexin57 occur in very close proximity to ribbon synapses formed by rod photoreceptors, as well as in close proximity to Cx36-containing gap junctions between rods and cones.     

Identification of connexin36 in gap junctions between neurons in rodent locus coeruleus

Locus coeruleus neurons are strongly coupled during early postnatal development, and it has been proposed that these neurons are linked by extraordinarily abundant gap junctions consisting of connexin32 (Cx32) and connexin26 (Cx26), and that those same connexins abundantly link neurons to astrocytes. Based on the controversial nature of those claims, immunofluorescence imaging and freeze-fracture replica immunogold labeling were used to re-investigate the abundance and connexin composition of neuronal and glial gap junctions in developing and adult rat and mouse locus coeruleus. In early postnatal development, connexin36 (Cx36) and connexin43 (Cx43) immunofluorescent puncta were densely distributed in the locus coeruleus, whereas Cx32 and Cx26 were not detected. By freeze-fracture replica immunogold labeling, Cx36 was found in ultrastructurally-defined neuronal gap junctions, whereas Cx32 and Cx26 were not detected in neurons and only rarely detected in glia. In 28-day postnatal (adult) rat locus coeruleus, immunofluorescence labeling for Cx26 was always co-localized with the glial gap junction marker Cx43; Cx32 was associated with the oligodendrocyte marker 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNPase); and Cx36 was never co-localized with Cx26, Cx32 or Cx43. Ultrastructurally, Cx36 was localized to gap junctions between neurons, whereas Cx32 was detected only in oligodendrocyte gap junctions; and Cx26 was found only rarely in astrocyte junctions but abundantly in pia mater. Thus, in developing and adult locus coeruleus, neuronal gap junctions contain Cx36 but do not contain detectable Cx32 or Cx26, suggesting that the locus coeruleus has the same cell-type specificity of connexin expression as observed ultrastructurally in other regions of the CNS. Moreover, in both developing and adult locus coeruleus, no evidence was found for gap junctions or connexins linking neurons with astrocytes or oligodendrocytes, indicating that neurons in this nucleus are not linked to the pan-glial syncytium by Cx32- or Cx26-containing gap junctions or by abundant free connexons composed of those connexins.     

A missense mutation in connexin26, D66H, causes mutilating keratoderma with sensorineural deafness (Vohwinkel's syndrome) in three unrelated families.

The multiplicity of functions served by intercellular gap junctions is reflected by the variety of phenotypes caused by mutations in the connexins of which they are composed. Mutations in the connexin26 (Cx26) gene ( GJB2 ) at 13q11-q13 are a major cause of autosomal recessive hearing loss (DFNB1), but have also been reported in autosomal dominant deafness (DFNA3). We now report a Cx26 mutation in three families with mutilating keratoderma and deafness [Vohwinkel's syndrome (VS; MIM 124500), as originally described]. VS is characterized by papular and honeycomb keratoderma associated with constrictions of digits leading to autoamputation, distinctive starfish-like acral keratoses and moderate degrees of deafness. In a large British pedigree, we have mapped the defect to the Cx26 locus. All 10 affected members were heterozygous for a non-conservative mutation, D66H, in Cx26. The same mutation was found subsequently in affected individuals from two unrelated Spanish and Italian pedigrees segregating VS, suggesting that D66H in Cx26 is a common mutation in classical VS. This mutation occurs at a highly conserved residue in the first extracellular domain of the Cx26 molecule, and may exert its effects by interfering with assembly into connexons, docking with adjacent cells or gating properties of the gap junction. Our results provide evidence that a specific mutation in Cx26 can impair epidermal differentiation, as well as inner ear function.     

An amino-terminal lysine residue of rat connexin40 that is required for spermine block

Spermine blocks connexin40 (Cx40) gap junctions, and two cytoplasmic amino-terminal domain glutamate residues are essential for this inhibitory activity. To further examine the molecular basis for block, we mutated a portion of a basic amino acid (HKH) motif on the Cx40 amino-terminal domain. Replacement of the Cx40 H15 + K16 residues with the Q15 + A16 sequence native to spermine-insensitive connexin43 (Cx43) gap junctions increased the equilibrium dissociation constant ( K _d) and reduced the maximum inhibition by spermine. The corresponding electrical distance (δ) approximation was decreased by about 50%. The transjunctional voltage ( V _j)-dependent gating of homotypic Cx40 H15Q + K16A mutant gap junctions was also significantly reduced. The minimum normalized steady-state junctional conductance ( G _min) increased from 0.17 to 0.72, with an increase in the half-inactivation voltage from 48 to 60 mV. However, the unitary junctional conductance (γ_j; 160 pS) was only slightly altered, and the relative cation/anion conductance and permeability ratios were unchanged from wild-type Cx40 gap junction channels. The relative K⁺/Cl⁻ permeability ( P _K/ P _Cl) ratio increased from six to ten when [KCl] was reduced to 25% of normal. These data suggest that the HKH motif at positions 15–17 is important to the conformational structure of the putative voltage sensor and spermine receptor of Cx40, without causing significant alteration of the electrostatic surface charge potentials that contribute to the ion selectivity of this gap junction channel.     

Replacement of connexin 43 by connexin 32 in a knock-in mice model attenuates aortic endothelium-derived hyperpolarizing factor-mediated relaxation

Previous studies demonstrated that intercellular communication through endothelial, smooth muscle or myoendothelial connexin channels contributes to the control of vascular tone. At least four connexin types are present in the arterial wall. The aim of the present work was to assess the role played by connexin 43 (Cx43)-formed gap junctions on vessel function. Aortic reactivity to noradrenaline, acetylcholine and sodium nitroprusside, and endothelium-derived hyperpolarizing factor (EDHF)-mediated relaxations, were analysed in a Cx43KI32 mouse model in which the coding region of Cx43 was replaced by that of connexin 32 (Cx32). Aortic rings were placed in organ baths containing a Krebs solution oxygenated at 37°C (pH 7.4). Confocal images of aortic rings confirmed connexin substitution in mutant mice. In control conditions, replacement of Cx43 by Cx32 in homozygous mutant mice did not modify endothelium-independent contractile responses to noradrenaline, or relaxations in response to sodium nitroprusside (endothelium independent) or acetylcholine (endothelium dependent). However, residual endothelium-dependent relaxations in response to acetylcholine after nitric oxide synthase and cyclooxygenase inhibition (EDHF type) were significantly reduced in homozygous Cx43KI32 mice (maximal effect values: 4.86 ± 0.37% of noradrenaline precontraction versus 7.06 ± 0.31% in wild-type, n = 8, P < 0.05). This attenuation was mimicked by treatment of rings from wild-type animals with the connexin-mimetic peptide ^37,43Gap27 (5 × 10⁻⁶ m ). In conclusion, replacement of Cx43 by Cx32 attenuates EDHF-mediated relaxations in mice aortic rings, suggesting that they are, at least in part, dependent on Cx43-formed gap junctions. In contrast, aortic responses to tested endothelium-independent agonists were not modified in knock-in animals.     

In vivo and in vitro expression of connexin 43 in human teeth

Gap junctions are composed of transmembrane proteins belonging to the connexin family. These proteins permit the exchange of mall regulatory molecules directly between cells for the control of growth, development and differentiation. Although the presence of gap junctions in teeth has been already evidenced, the involved connexins have not yet been identified in human species. Here, we examined the distribution of connexin 43 (Cx43) in embryonic and permanent intact and carious human teeth. During tooth development, Cx43 localized both in epithelial and mesenchymal dental cells, correlated with cytodifferentiation gradients. In adult intact teeth, Cx43 was distributed in odontoblast processes. While Cx43 expression was downregulated in mature intact teeth, Cx43 appeared to be upregulated in odontoblasts facing carious lesions. In cultured pulp cells, Cx43 expression was related to the formation of mineralized nodules. These results indicate that Cx43 expression is developmentally regulated in human dental tissues, and suggest that Cx43 may participate in the processes of dentin formation and pathology.     

Dilated bile canaliculi and attenuated decrease of nerve-dependent bile secretion in connexin32-deficient mouse liver

Gap junction channels in the rodent liver are composed of connexin26 (Cx26) and connexin32 (Cx32) proteins. Gap junctional intercellular communication in the mouse liver enhances the effects of hormonal or sympathetic stimulation of glucose release from glycogen stores. To determine whether contraction of bile canaliculi and bile secretion are dependent on the function of gap junction channels, we compared wild-type and connexin32-deficient mice. Confocal laser scanning microscopy of the wild-type mouse liver confirmed the close association of connexin26 and -32 proteins with the zona occludens-1 protein and actin filaments of the bile canaliculi. The decrease of bile flow after electrical stimulation of sympathetic nerves in the perfused liver was attenuated in the Cx32-deficient liver compared with wild-type controls. The amount of secreted bile, however, was similar in wild-type and Cx32-deficient livers. Furthermore, Cx32-deficient mice exhibited dilated bile canaliculi, suggesting that the contraction of bile canaliculi could be impaired in these animals.     

Characterization of connexin 30.3 and 43 in thymocytes

During maturation, thymocytes interact directly and indirectly with different cell types of the thymic microenvironment. Such a cellular communication has been basically ascribed to soluble factors and surface receptors. However, little attention has been given to cellular communication mediated by gap junctions. The existence of these intercellular channels in the immune system remained a controversial issue since the 1970s until recently, when a growing body of evidence has indicated their presence and physiological roles in the immune system. In this work, we investigated whether thymocytes express gap junction-forming proteins (connexins, Cx) and are capable of forming functional intercellular channels. Using RT-PCR, we demonstrated that thymocytes express the mRNA for two Cx isoforms: Cx30.3 and Cx43, but not for Cx26, Cx30, Cx31, Cx31.1, Cx32, Cx33, Cx36, Cx37, Cx40, Cx45, Cx46, and Cx50. In addition, the presence of Cx30.3 and Cx43 was confirmed using different techniques (RNase protection assay, western blot and immunofluorescence). However, despite the expression of these two Cxs, we did not detect functional homocellular coupling between thymocytes or between EL-4 cells (a Cx43 expressing thymic lymphoma-derived cell line) or heterocellular coupling between thymocytes and thymic epithelial cells (TEC) or between EL-4 and TEC in unstimulated conditions. Concluding, in this study, we described for the first time the expression of connexins in thymocytes, which may constitute a new molecule having a functional role in thymocytes maturation.     

The connexin26 S17F mouse mutant represents a model for the human hereditary keratitis-ichthyosis-deafness syndrome

Mutations in the GJB2 gene coding for connexin26 (Cx26) can cause a variety of deafness and hereditary hyperproliferative skin disorders in humans. In this study, we investigated the Cx26S17F mutation in mice, which had been identified to cause the keratitis-ichthyosis-deafness (KID) syndrome in humans. The KID syndrome is characterized by keratitis and chronic progressive corneal neovascularization, skin hyperplasia, sensorineural hearing loss and increased carcinogenic potential. We have generated a conditional mouse mutant, in which the floxed wild-type Cx26-coding DNA can be deleted and the Cx26S17F mutation is expressed under control of the endogenous Cx26 promoter. Homozygous mutants are not viable, whereas the surviving heterozygous mice show hyperplasia of tail and foot epidermis, wounded tails and annular tail restrictions, and are smaller than their wild-type littermates. Analyses of auditory brainstem responses (ABRs) indicate an ～35 dB increased hearing threshold in these mice, which is likely due to the reduction of the endocochlear potential by 20-40%. Our results indicate that the Cx26S17F protein, which does not form functional gap junction channels or hemichannels, alters epidermal proliferation and differentiation in the heterozygous state. In the inner ear, reduced intercellular coupling by heteromeric channels composed of Cx26S17F and Cx30 could contribute to hearing impairment in heterozygous mice, while remaining wild-type Cx26 may be sufficient to stabilize Cx30 and partially maintain cochlear homeostasis. The phenotype of heterozygous mice resembles many of the symptoms of the human KID syndrome. Thus, these mice represent an appropriate model to further investigate the disease mechanism.     

Mutations in GJA1 (connexin 43) are associated with non-syndromic autosomal recessive deafness.

Mutations in four members of the connexin gene family have been shown to underlie distinct genetic forms of deafness, including GJB2 [connexin 26 (Cx26)], GJB3 (Cx31), GJB6 (Cx30) and GJB1 (Cx32). We have found that alterations in a fifth member of this family, GJA1 (Cx43), appear to cause a common form of deafness in African Americans. We identified two different GJA1 mutations in four of 26 African American probands. Three were homozygous for a Leu-->Phe substitution in the absolutely conserved codon 11, whereas the other was homozygous for a Val-->Ala transversion at the highly conserved codon 24. Neither mutation was detected in DNA from 100 control subjects without deafness. Cx43 is expressed in the cochlea, as is demonstrated by PCR amplification from human fetal cochlear cDNA and by RT-PCR of mouse cochlear tissues. Immunohistochemical staining of mouse cochlear preparations showed immunostaining for Cx43 in non-sensory epithelial cells and in fibrocytes of the spiral ligament and the spiral limbus. To our knowledge this is the first alpha connexin gene to be associated with non-syndromic deafness. Cx43 must also play a critical role in the physiology of hearing, presumably by participating in the recycling of potassium to the cochlear endolymph.     

Cx31 and Cx43 double-deficient mice reveal independent functions in murine placental and skin development.

The overlapping expression of gap junctional connexins in tissues has indicated that the channels may compensate for each other. During development, Cx31 and Cx43 are coexpressed in preimplantation embryos, in the spongiotrophoblast of the placenta and in the epidermis. This study shows that Cx31/Cx43 double-deficient mice exhibit the known phenotypes of the single-knockout strains but no combined effects. Thus, Cx43, coexpressed with Cx31 at midgestation in the spongiotrophoblast of the placenta, cannot be responsible for a partial rescue of the lethal Cx31 knockout phenotype, as assumed before (Plum et al. [2001] Dev Biol 231:334-337). It follows that both connexins have unique functions in placental development. Despite an altered expression of other epidermal connexin mRNAs, epidermal differentiation and physiology was unaltered by the absence of Cx31 and Cx43. Therefore, in epidermal and preimplantation development, gap junctional communication can probably be compensated by other isoforms coexpressed with Cx31 and Cx43.     

A single evoked afterdischarge produces rapid time-dependent changes in connexin36 protein expression in adult rat dorsal hippocampus

Gap junctions between neurons contribute to synchronous neuronal firing and may play a role in the pathophysiology of epilepsy. We examined the expression of a number of gap junction subunits, including the neuronal gap junction forming protein connexin36 (Cx36), in the hippocampus at various time points following an electrically stimulated afterdischarge (AD) in freely-moving animals. Once recovered from electrode implantation, animals were tested with an escalating series of stimulations until an AD was evoked. Suprathreshold stimulation produced a brief AD with no convulsion. Groups of animals were sacrificed at 3, 12, and 24h post-stimulation, and connexin expression was assessed via semiquantitative immunoblotting. Compared to implanted non-stimulated controls, a significant decrease in Cx36 expression was observed in the stimulated dorsal hippocampus at 3h post-stimulation, which returned to control levels by 24h. No changes were seen in the ventral hippocampus. As well, no changes were seen in other selected connexin proteins including Cx26, Cx32, and Cx43, thought to be expressed primarily in glia, in either dorsal or ventral hippocampus. These data suggest that a relatively brief hypersynchronous neuronal discharge can produce rapid and specific changes in Cx36 expression, which may have implications for both normal brain function and the pathophysiology of epilepsy.