Global ischemia-induced increases in the gap junctional proteins connexin 32 (Cx32) and Cx36 in hippocampus and enhanced vulnerability of Cx32 knock-out mice

Abstract

Gap junctions are conductive channels that connect the interiors of coupled cells. In the hippocampus, GABA-containing hippocampal interneurons are interconnected by gap junctions, which mediate electrical coupling and synchronous firing and thereby promote inhibitory transmission. The present study was undertaken to examine the hypothesis that the gap junctional proteins connexin 32 (Cx32; expressed by oligodendrocytes, interneurons, or both), Cx36 (expressed by interneurons), and Cx43 (expressed by astrocytes) play a role in defining cell-specific patterns of neuronal death in hippocampus after global ischemia in mice. Global ischemia did not significantly alter Cx32 and Cx36 mRNA expression and slightly increased Cx43 mRNA expression in the vulnerable CA1, as assessed by Northern blot analysis and in situ hybridization. Global ischemia induced a selective increase in Cx32 and Cx36 but not Cx43 protein abundance in CA1 before onset of neuronal death, as assessed by Western blot analysis. The increase in Cx32 and Cx36 expression was intense and specific to parvalbumin-positive inhibitory interneurons of CA1, as assessed by double immunofluorescence. Protein abundance was unchanged in CA3 and dentate gyrus. The finding of increase in connexin protein without increase in mRNA suggests regulation of Cx32 and Cx36 expression at the translational or post-translational level. Cx32(Y/-) null mice exhibited enhanced vulnerability to brief ischemic insults, consistent with a role for Cx32 gap junctions in neuronal survival. These findings suggest that Cx32 and Cx36 gap junctions may contribute to the survival and resistance of GABAergic interneurons, thereby defining cell-specific patterns of global ischemia-induced neuronal death.

Fig. 1.

Histology at 72 hr after sham operation and 72 hr and 7 d after global ischemia in mice Toluidine blue labeling of coronal brain sections at the level of the dorsal hippocampus from control mice (n = 3; A, D) and experimental mice at 72 hr after ischemia (n = 3;B, E) revealed no detectable neuronal damage. At 7 d after ischemia (n = 3), cell loss was observed, primarily in CA1 (C, F). Brief (20 min) global ischemia was induced by bilateral occlusion of the common carotid arteries as described in Materials and Methods. Scale bars, 50 μm.so, Stratum oriens; sr, stratum radiatum.

Fig. 2.

Patterns of global ischemia-induced alterations in Cx32, Cx36, and Cx43 mRNA expression in mouse hippocampus, as assessed by in situ hybridization of coronal brain sections.A–C, In control animals, Cx32, Cx36, and Cx43 mRNA expression was prominent in the pyramidal cell layers of the hippocampal CA1 and CA3 and in the granule cell layer of dentate gyrus (DG). Global ischemia induced a marked increase in Cx43 (C) mRNA expression in CA1 (closed circles). Cx32 (A) and Cx36 (B) were unchanged at all times examined in CA1. Connexin mRNA expression was unchanged in the dentate gyrus except for Cx36 (open circles). D, Global ischemia also downregulated mRNA encoding the AMPAR subunit GluR2 in CA1.E, GluR1 mRNA expression was unchanged in all subfields as late as 48 hr; GluR1 was modestly reduced at 72 hr, presumably because of onset of cell loss. Data shown are mean densities ± SEM of autoradiographic films for in situ hybridization of Cx32, Cx36, and Cx43 mRNAs in the hippocampal CA1 at 2, 8, 24, 48, and 72 hr after the ischemic insult.

Fig. 3.

Global ischemia-induced downregulation of Cx32 mRNA expression in mouse hippocampus, as assessed by Northern blot analysis. A, Representative film autoradiograms of Northern blots probed with highly specific ³⁵S-labeled RNA probes directed to the Cx32, Cx36, and Cx43 mRNAs. Samples of total RNA extracts isolated from control animals at 24 hr after sham operation (n = 3) and experimental animals at 24 hr after ischemia (n = 3) were subjected to Northern blot analysis as described in Materials and Methods. Global ischemia induced a significant downregulation in Cx32 mRNA. B, Quantitation of Northern blot analysis data. Means ± SEM of band densities are shown from three independent experiments involving samples from three experimental and three control animals. Band densities for samples from experimental animals were normalized to the corresponding band densities for samples from control animals.

Fig. 4.

Global ischemia-induced changes in connexin subunit abundance in hippocampal CA1. Global ischemia induced an increase in Cx32 and Cx36 protein abundance in CA1 but did not detectably alter Cx43 protein abundance. Film autoradiograms (top panels) and band densities (bottom panels) of Western blots probed for Cx32 (A), Cx36 (B), Cx43 (C), and GluR2 (D) protein are shown. Samples of protein extracts from the CA1 of control animals at 24 hr after sham operation (n = 3) and experimental animals at 8, 24, 48, and 72 hr after ischemia (n = 3 for each treatment group at each time point) were subjected to Western blot analysis as described in Materials and Methods. Means ± SEM of band densities were from three independent experiments. Band densities for samples from experimental animals were normalized to those for control animals. Global ischemia induced a marked upregulation in Cx32 and Cx36 and a downregulation in GluR2 protein abundance in CA1 after ischemia. Cx43 protein abundance was unchanged at all times examined after global ischemia.

Fig. 5.

Global ischemia markedly increases Cx32 protein expression in parvalbumin-positive inhibitory interneurons in CA1. Cx32 (A, D, G, J), PV (B, E, H, K), and merged immunofluorescence images (C, F, I, L) show inhibitory interneurons within the CA1 pyramidal cell layer in brain sections from control animals (A–C) at 24 hr after sham operation and experimental animals at 24 hr (D–F), 48 hr (G–I) and 7 d (J–L) after global ischemia. Cx32 immunofluorescence in cell bodies exhibited a high coincidence with PV immunofluorescence (C, F, I, L) throughout the CA1 pyramidal cell layer. Cx32 immunofluorescence exhibited intense puncta along the dendrites of PV-positive inhibitory interneurons. Global ischemia markedly increased Cx32 immunofluorescence in parvalbumin-positive inhibitory interneurons in the CA1 pyramidal layer at 24 and 48 hr with some decline at 72 hr. Data are typical of 10 sections per animal from a minimum of three animals per time point and treatment group. Cx32 immunofluorescence is visualized in green, and PV immunofluorescence is visualized in red. so, Stratum oriens; sp, stratum pyramidale; sr, stratum radiatum. Scale bar, 50 μm.

Fig. 6.

Global ischemia markedly increases Cx36 immunolabeling in CA1. Cx36 (A, D, G,J), PV (B, E, H,K), and merged immunofluorescence images (C,F, I, L) show Cx36 immunoreactivity and inhibitory interneurons in the CA1 pyramidal cell layer in brain sections from control animals (A–C) at 24 hr after sham operation and experimental animals at 24 (D–F), 48 (G–I), and 72 (J–L) hr after global ischemia. Global ischemia markedly increased punctate Cx36 immunolabeling in the pyramidal cell layer extending out into the stratum oriens and stratum radiatum at 24 and 48 hr. There was Cx36 labeling of PV-positive inhibitory neurons at 72 hr. Data are typical of a minimum of 10 sections per animal from three animals per time point and treatment group. Cx36 immunofluorescence is visualized in green, and PV immunofluorescence is visualized in red.so, Stratum oriens; sp, stratum pyramidale; sr, stratum radiatum. Scale bar, 50 μm.

Fig. 7.

Cx32(Y/−) null mice exhibit enhanced vulnerability to global ischemia-induced neurodegeneration.A, Southern blot analysis of mice cDNAs. RT-PCR was performed on DNA samples obtained from tail biopsies of Cx32 wild-type and deficient mice and subjected to Southern blot hybridization.First lane, Wild-type; second lane, heterozygous female Cx32(+/−); third lane, homozygous Cx32(Y/−) null mice. The 881-bp amplicon indicates the presence of the wild-type Cx32 gene; the 441-bp amplicon indicates the presence of the Cx32 defective allele. B, Cx32(Y/−) null mice exhibit enhanced vulnerability to global ischemia-induced neurodegeneration. Toluidine blue staining of coronal brain sections shows the dorsal hippocampus of wild-type and Cx32(Y/−) null mice. Animals were killed 7 d after sublethal (brief, 10 min) global ischemia or sham operation. At 7 d after brief ischemia, wild-type (WT) mice exhibited no detectable neuronal loss; Cx32 null mice exhibited a range of neuronal loss. Scale bar:first and third columns, 100 μm;second and fourth columns, 50 μm.DG, Dentate gyrus; so, stratum oriens;sr, stratum radiatum. C, Quantitation of ischemia-induced damage. Neuronal damage scores are shown for Cx32(Y/−) null mice (left) and Cx32(Y/+) wild-type mice (right). Neuronal damage was assessed as follows: 0, no neuronal damage; 1, minor neuronal damage (<30% neurons are dead); 2, moderate neuronal damage (30–70% of neurons are dead); 3, severe (>70% neurons are dead); scores from right and left sides were summed for each animal's total score. *p < 0.01 for Cx32(Y/−) null mice versus Cx32(Y/+) wild-type mice.