Barbara Teubner

The murine gap junction gene connexin36 is highly expressed in mouse retina and regulated during brain development.

A new gap junction gene isolated from rat brain cDNA, mouse retina cDNA and mouse genomic DNA is called connexin36, since it codes for a connexin protein of 321 amino acids corresponding to the theoretical molecular mass of 36 045 kDa (rat) and 36 084 kDa (mouse). Only one amino acid residue differs between rat and mouse connexin36. In the single murine connexin36 gene, an 1.14‐kb intron interrupts the coding region, similar as in the homologous skate connexin35 gene. Because of this unique feature, mouse connexin36 differs from the other 13 murine connexin genes and is suggested to form a new δ subclass of connexins. Connexin36 mRNA (2.9 kb) is highly expressed in adult retina and less abundant in brain where it gradually increased during fetal development until day 7 post partum, and decreased thereafter.

Altered connexin expression and wound healing in the epidermis of connexin-deficient mice.

To analyze the effect of connexin loss on the repair of wounded tail skin, we have studied the following transgenic mouse mutants: connexin30–/–, connexin31–/– and connexin43Cre-ER(T)/fl (for inducible deletion of the connexin43 coding region). Connexin43 and connexin31 are expressed in the basal and spinous layers of wild-type epidermis, whereas connexin31 and small amounts of connexin30, as well as connexin26 proteins, were found in the granulous layer. Connexin43 was downregulated in connexin31-deficient mice, whereas mice with reduced connexin43 exhibited an upregulation of connexin30. During wound healing, connexin30 and connexin26 proteins were upregulated in all epidermal layers, whereas connexin43 and connexin31 protein expression were downregulated. In connexin31–/– mice, reduced levels of connexin30 protein were observed on days 1 and 2 after wounding. The closure of epidermal wounds in mice with decreased amounts of connexin43 protein occurred one day earlier. Under these conditions the expression profiles of connexin30 and connexin31 were also temporarily shifted by one day. Furthermore, dye transfer between keratinocytes in skin sections from connexin43-deficient mice was decreased by 40%. These results suggest that downregulation of connexin43 appears to be a prerequisite for the coordinated proliferation and mobilization of keratinocytes during wound healing.

Expression patterns of connexin genes in mouse retina

To analyze the molecular basis of gap junctional communication in mouse retina, we examined the expression pattern of the following 13 connexin (Cx) genes: Cx26, Cx30, Cx30.3, Cx31, Cx31.1, Cx32, Cx36, Cx37, Cx40, Cx43, Cx45, Cx46, and Cx50. By using reverse transcriptase‐polymerase chain reactions with primer oligonucleotides to murine connexin genes, we detected mRNAs of Cx26, Cx31, Cx32, Cx36, Cx37, Cx40, Cx43, Cx45, and Cx50. Retinae from heterozygous mice with targeted replacement of most of the Cx45 open reading frame by a lacZ reporter gene showed Cx45 promoter activity in somata of the ganglion cell layer and the inner nuclear layer. Immunoblot and immunofluorescence analyses with antibodies generated to murine connexin epitopes revealed the presence of Cx36, Cx37, Cx43, and Cx45 proteins: The outer and inner plexiform layer were immunopositive for Cx36 and Cx45. Cx37 immunoreactivity was found in blood vessels of the inner retina. Cx43 immunolabeling was detected in the ganglion cell layer and nerve fiber layer where it was largely colocalized with immunostaining of glial fibrillary acidic protein suggesting that Cx43‐positive cells could be of glial origin. No Cx26 protein was detected in retina by using Cx26 antibodies for immunoblot analyses or confocal microscopy. Furthermore, comparative immunofluorescence analyses of retinae from mice deficient for Cx31, Cx32, or Cx40 with retinae of wild‐type mice revealed no specific immunostaining. Our results demonstrate regional specificity in expression of connexin genes in mouse retina and, thus, provide a basis for future assignments of functional defects in connexin‐deficient mice to cells in different regions of the retina. J. Comp. Neurol. 425:193–201, 2000.

Mice with astrocyte-directed inactivation of connexin43 exhibit increased exploratory behaviour, impaired motor capacities, and changes in brain acetylcholine levels

Gap junctions mediate communication between many cell types in the brain. Gap junction channels are composed of membrane‐spanning connexin (Cx) proteins, allowing the cell‐to‐cell passage of small ions and metabolites. Cx43 is the main constituent of the brain‐spanning astrocytic gap junctional network, controlling activity‐related changes in ion and glutamate concentrations as well as metabolic processes. In astrocytes, deletion of Cx43‐coding DNA led to attenuated gap junctional coupling and impaired propagation of calcium waves, known to influence neuronal activity. Investigation of the role of Cx43 in behaviour has been impossible so far, due to postnatal lethality of its general deletion. Recently, we have shown that deletion of Cx30, which is also expressed by astrocytes, affects exploration, emotionality, and neurochemistry in the mouse. In the present study, we investigated the effects of the astrocyte‐directed inactivation of Cx43 on mouse behaviour and brain neurochemistry. Deletion of Cx43 in astrocytes increased exploratory activity without influencing habituation. In the open field, but not in the elevated plus‐maze, an anxiolytic‐like effect was observed. Rotarod performance was initially impaired, but reached control level after further training. In the water maze, Cx43 deficient mice showed a steeper learning course, although final performance was similar between groups. Cx43 inactivation in astrocytes increased acetylcholine content in the frontal cortex of water maze‐trained animals. Results are discussed in terms of altered communication between astrocytes and neurons, possible compensation processes, and differential effects of Cx30‐ and astrocyte‐specific Cx43 deletion.

Connexin30‐deficient mice show increased emotionality and decreased rearing activity in the open‐field along with neurochemical changes

Gap‐junction channels in the brain, formed by connexin (Cx) proteins with a distinct regional/cell‐type distribution, allow intercellular electrical and metabolic communication. In astrocytes, mainly the connexins 43, 26 and 30 are expressed. In addition, connexin30 is expressed in ependymal and leptomeningeal cells, as well as in skin and cochlea. The functional implications of the astrocytic gap‐junctional network are not well understood and evidence regarding their behavioural relevance is lacking. Thus, we have tested groups of Cx30−/−, Cx30+/−, and Cx30+/+ mice in the open‐field, an object exploration task, in the graded anxiety test and on the rotarod. The Cx30−/− mice showed reduced exploratory activity in terms of rearings but not locomotion in the open‐field and object exploration task. Furthermore, Cx30−/− mice exhibited anxiogenic behaviour as shown by higher open‐field centre avoidance and corner preference. Graded anxiety test and rotarod performance was similar across groups. The Cx30−/− mice had elevated choline levels in the ventral striatum, possibly related to their aberrant behavioural phenotypes. The Cx30+/− mice had lower dopamine and metabolite levels in the amygdala and ventral striatum and lower hippocampal 5‐hydroxyindole acid (5‐HIAA) concentrations relative to Cx30+/+ mice. Furthermore, the Cx30+/− mice had lower acetylcholine concentrations in the ventral striatum and higher choline levels in the neostriatum, relative to Cx30+/+ mice. Our data suggest that the elimination of connexin30 can alter the reactivity to novel environments, pointing to the importance of gap‐junctional signalling in behavioural processes.

Expression of connexin genes in hippocampus of kainate-treated and kindled rats under conditions of experimental epilepsy

We have analyzed whether the expression of connexin genes is altered in the hippocampus of kindled and kainate-treated rats, i.e., animal models of human temporal lobe epilepsy. We have tested this hypothesis by analyzing mRNA, protein abundance and cellular location of connexins (Cx) 43, 36, 32 and 30. The expression of glial fibrillary acid protein and mRNA was also monitored both in kainate-treated and kindled rats, in order to take into account reactive gliosis under these conditions. We found significantly increased expression of GFAP mRNA (100%) and protein (178%) in kainate-treated rats 4 weeks after kainate application, whereas in kindled rats only moderate increases of GFAP mRNA and protein were detected 2-3 weeks (group 2) or 4-6 weeks (group 1) after the last stage 5 induced seizure. Under gliotic conditions, connexins 43 and 30 mRNA or protein expression in astrocytes of kainate-treated rats were nearly unaffected. Cx36 mRNA expression (presumably in neurons) was significantly reduced (44%), whereas abundance of Cx36 protein was only slightly reduced. In both groups of kindled rats, Cx30 and Cx43 mRNA or protein expression were either slightly decreased or unchanged. Again, Cx36 mRNA and protein expression were reduced by about half in group 2. Immunofluorescence analysis of Cx43, Cx36 and Cx30 expression revealed that 4 weeks after the last kainate administration or kindling, cellular localization of these connexins was indistinguishable from control animals.

Immunohistochemical detection of the neuronal connexin36 in the mouse central nervous system in comparison to connexin36-deficient tissues

Abstract. Investigating the spatial and temporal expression of connexin36 (Cx36) protein in neuronal tissue is of prime importance to understand the molecular mechanisms underlying extensive electrical coupling. Although Cx36 mRNA was shown to be expressed in neurons of the central nervous system in different studies, only the determination of Cx36 protein expression allows a correlation between localization and its functional role in gap junction-mediated neuronal coupling. After the initial use of antibodies recognizing the skate connexin35 protein, antibodies directed to the mammalian Cx36 sequence allowed the detailed investigation of Cx36 cellular localization. However, results on Cx36 protein distribution still remained controversial in some areas of the central nervous system. In the present study, we have investigated: (a) the distribution of Cx36 protein in various areas of the central nervous system and (b) determined the specificity in the immunohistochemical staining of two polyclonal antibodies comparing wildtype and Cx36-deficient mice. In some areas of the central nervous system, for example in the retina and the inferior nuclear olivary complex, Cx36 antibodies were highly specific, and in the cerebellar cortex, Cx36 protein expression was partly specific. In other regions, particularly in pyramidal cells of the hippocampal formation, non-specific staining was prevalent, indicating that Cx36 antibodies also recognize proteins other than Cx36 in these tissues. The present results argue for a re-evaluation of many documented immunohistochemical protein distribution patterns and require, not only in connexin research, their assessment using null-mutant animals.