Loss of connexin36 channels alters beta-cell coupling, islet synchronization of glucose-induced Ca2+ and insulin oscillations, and basal insulin release.

Normal insulin secretion requires the coordinated functioning of beta-cells within pancreatic islets. This coordination depends on a communications network that involves the interaction of beta-cells with extracellular signals and neighboring cells. In particular, adjacent beta-cells are coupled via channels made of connexin36 (Cx36). To assess the function of this protein, we investigated islets of transgenic mice in which the Cx36 gene was disrupted by homologous recombination. We observed that compared with wild-type and heterozygous littermates that expressed Cx36 and behaved as nontransgenic controls, mice homozygous for the Cx36 deletion (Cx36(-/-)) featured beta-cells devoid of gap junctions and failing to exchange microinjected Lucifer yellow. During glucose stimulation, islets of Cx36(-/-) mice did not display the regular oscillations of intracellular calcium concentrations ([Ca(2+)](i)) seen in controls due to the loss of cell-to-cell synchronization of [Ca(2+)](i) changes. The same islets did not release insulin in a pulsatile fashion, even though the overall output of the hormone in response to glucose stimulation was normal. However, under nonstimulatory conditions, islets lacking Cx36 showed increased basal release of insulin. These data show that Cx36-dependent signaling is essential for the proper functioning of beta-cells, particularly for the pulsatility of [Ca(2+)](i) and insulin secretion during glucose stimulation.

Reduction of high-frequency network oscillations (ripples) and pathological network discharges in hippocampal slices from connexin 36-deficient mice.

Recent evidence suggests that electrotonic coupling is an important mechanism for neuronal synchronisation in the mammalian cortex and hippocampus. Various types of network oscillations have been shown to depend on, or be sharpened by, gap junctions between inhibitory interneurones or excitatory projection cells. Here we made use of a targeted disruption of the gene coding for Cx36, a recently discovered neuronal gap junction subunit, to analyse its role in hippocampal network behaviour. Mice lacking Cx36 are viable and lack obvious morphological or behavioural abnormalities. Stimulation of afferent and efferent fibre pathways in hippocampal slices revealed a largely normal function of the synaptic circuitry, including tetanically evoked network oscillations. Spontaneous sharp waves and ripple (approximately 200 Hz) oscillations, however, occurred less frequently in slices from Cx36 -/- mice, and ripples were slightly slower than in littermate controls. Moreover, epileptiform discharges elicited by 4-aminopyridine were attenuated in slices from Cx36 -/- mice. Our findings indicate that Cx36 plays a role in the generation of certain forms of network synchronisation in the hippocampus, namely sharp wave-ripple complexes and hypersynchronous epileptiform discharges.

Structural and functional diversity of connexin genes in the mouse and human genome.

Gap junctions are clustered channels between contacting cells through which direct intercellular communication via diffusion of ions and metabolites can occur. Two hemichannels, each built up of six connexin protein subunits in the plasma membrane of adjacent cells, can dock to each other to form conduits between cells. We have recently screened mouse and human genomic data bases and have found 19 connexin (Cx) genes in the mouse genome and 20 connexin genes in the human genome. One mouse connexin gene and two human connexin genes do not appear to have orthologs in the other genome. With three exceptions, the characterized connexin genes comprise two exons whereby the complete reading frame is located on the second exon. Targeted ablation of eleven mouse connexin genes revealed basic insights into the functional diversity of the connexin gene family. In addition, the phenotypes of human genetic disorders caused by mutated connexin genes further complement our understanding of connexin functions in the human organism. In this review we compare currently identified connexin genes in both the mouse and human genome and discuss the functions of gap junctions deduced from targeted mouse mutants and human genetic disorders.

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

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.