Deletion of astrocyte connexins 43 and 30 leads to a dysmyelinating phenotype and hippocampal CA1 vacuolation

Abstract

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.

Figure 1.

Gray and white matter pathology in H&E-stained sagittal brain sections of 6-month-old dKO mice. A–C, Low-power views of mGFAP-Cre (A), Cx43^del Cx30^+/− (B), and dKO (C) mice show vacuolation in the corpus callosum and CA1 region of hippocampus in dKO mice only. D, E, High-power views of CA1 in mGFAP-Cre and dKO mice. F, Percentage area white space in CA1 is significantly greater (p < 0.01) in dKO compared with either Cx43^del Cx30^+/− or mGFAP-Cre mice. G–I, Low-power views of cerebellum in the same animals as above show vacuolation in cerebellar white matter in dKO mice. J, K, High-power views of cerebellar white matter from mGFAP-Cre and dKO mice. L, Percentage area of white space in deep cerebellar white matter is significantly greater (p < 0.05) in dKO compared with either Cx43^del Cx30^+/− or mGFAP-Cre mice. Scale bars are 100 and 10 μm, respectively, for low- and high-power figures.

Figure 2.

Onset of vacuolation and deletion of Cx43 occurs approximately at postnatal day 23 and persists into adulthood. A–E, H&E-stained sagittal brain sections through the hippocampus and corpus callosum of dKO and non-Cre (Cx43^fl/fl Cx30^−/−) littermates at P4 (A), P15 (B), P23 (C), 2 months (D) and 12 months (E). F–I, Western blots of dKO and non-Cre brain, cerebellum, and spinal cord at P4 (F), P15 (G), P23 (H), and P30 (I) demonstrate that Cx43 expression becomes reduced in dKO pups between P15 and P23. Western blots were stripped and stained for GFAP. mos, Months; Sp., spinal. Scale bar: (A), 400 μm; (B–E), 200 μm.

Figure 3.

Pathology in dKO animals is associated with astrogliosis in gray and white matter. A–C, Low-power views of GFAP immunostaining in hippocampus and corpus callosum in mGFAP-Cre (A), Cx43^del Cx30^+/− (B), and dKO (C) mice. D, E, Higher-power views of boxed region in C illustrating GFAP immunoreactivity in association with vacuolated areas of CA1. F, Percentage area GFAP immunoreactivity in the CA1 region of the hippocampus is significantly greater (p < 0.01) in dKO versus control (mGFAP-Cre and Cx43^del Cx30^+/−) mice. G, H, Higher-power views of vacuolated areas of the corpus callosum (CC) in dKO mice. I, Percentage area GFAP immunoreactivity in the corpus callosum was significantly greater (p < 0.05) in dKO versus control (mGFAP-Cre and Cx43^del Cx30^+/−) mice. J–L, Low-power views of GFAP immunoreactivity in cerebellum of mGFAP-Cre (J), Cx43^del Cx30^+/− (K), and dKO (L) mice. M, N, Higher-power views of vacuolated areas of the cerebellar white matter in dKO mice. O, Percentage area GFAP immunoreactivity in the deep cerebellar white matter was significantly greater (p < 0.01) in dKO versus control (mGFAP-Cre and Cx43^del Cx30^+/−) mice. Scale bar: (A–C, J–L), 200 μm; (D, G, M), 50 μm; (E, H, N), 10 μm.

Figure 4.

Electron micrographs depicting oligodendrocyte vacuolation within dKO white matter. A, Astrocyte (As) and oligodendrocyte (olig) in white matter of wild-type spinal cord. The astrocyte can be identified by marginal chromatin and cytoplasmic intermediate filaments. B–G, Vacuolated oligodendrocytes and astrocyte in dKO white matter. B, Vacuolated oligodendrocyte abutting edematous astrocyte in dKO cerebellar white matter. The oligodendrocyte is identified by marginal chromatin, microtubules, and relatively electron-dense cytoplasm. Note that the astrocyte also shows extensive pathology with loss of well defined cytoplasmic organelles. The oligodendrocyte is outlined in red. C, Highly compartmentalized oligodendrocyte cytoplasm. D, Higher-power view of the field in C helps identify the compartmentalized cytoplasm as oligodendrocytic due to the presence of microtubules and lack of filaments, and because its membranes are contiguous with compact myelin. E, An oligodendrocyte containing numerous membrane-bound vacuoles within its cytoplasm. The oligodendrocyte is outlined in red. Inset: immunohistochemical staining showing the nucleus of a vacuolated cell positive for both DAPI (blue) and olig2 (red). Note the unaffected, olig2-negative nucleus at right (arrow). F, Detail from E showing a continuous ribbon of oligodendrocyte cytoplasm connecting the perinuclear cytoplasm and plasma membrane between two vacuoles, demonstrating that the vacuoles are contained within the oligodendrocyte. G, Intramyelinic edema. H, I, dKO and wild-type g-ratios (H) and axon diameter (I) are not significantly different in the cerebellum. Magnification: (A), 4000×; (B), 2600×; (C), 6000×; (D), 35,000×; (E), 5000×; (F), 21,000×; (G), 3600×.

Figure 5.

Myelin pathology in dKO white matter. A–C, Toluidine blue stained 1 μm epoxy sections of the dorsal cuneate of the lumbar spinal cord of non-Cre (Cx43^fl/fl Cx30^−/− (A) and dKO (B, C) mice. D, Electron micrograph showing enlarged oligodendrocyte cytoplasm interpolating between an apparently normal axon and its myelin sheath. Inset depicts ectopic expression of myelin in one of several locations within the oligodendrocyte cytoplasm. E, Myelin whorls, likely representing Wallerian axonal degeneration. F, G, Edema and splitting of myelin sheaths. Scale bar: (A, B), 100 μm; (C), 10 μm. Magnification: (D), 4000×, inset: 15,000×; (E), 9000×; (F), 8000×; (G), 13,000×, inset: 4000×.

Figure 6.

Myelin and oligodendrocyte abnormalities are associated with sensorimotor deficits in dKO mice. A, Immunohistochemistry for Cx43 (red) and myelin basic protein (MBP; green) demonstrates Cx43 puncta adjacent to oligodendrocytes in an interfascicular chain (arrows) in the corpus callosum of a wild-type mouse. Blue, DAPI nuclear stain. B, C, Representative Western blot shows 40% reduced MBP in dKO corpus callosum homogenates relative to β-tubulin (p < 0.05). D–G, Immunohistochemistry for olig2 (red) and the mature marker CC1 (green) demonstrates that although total numbers of oligodendrocytes in control and dKO corpus callosum are similar, there are fewer mature oligodendrocytes in the dKO (p < 0.01). Asterisks in E mark white matter vacuoles. Blue, DAPI. H, dKO mice (×) fall off of the rotarod more quickly than do Cx43^fl/fl Cx30^−/− control mice (Cx30^−/−, ●) control mice. I, J, dKO mice (blue bars) have more slips (I) and are slower (J) than controls (black bars) while crossing wide and narrow balance beams. B–G, n = 5–6 mice per genotype. H–J, n = 3 mice per genotype.

Figure 7.

Astrocyte pathology in the CA1 pyramidal cell layer of the hippocampus and spatial memory impairment. A–C, Electron micrographs depicting edematous astrocyte (As) processes identified by the presence of glycogen granules and intermediate filaments within the CA1 region of the hippocampus of a 4-month-old dKO mouse. B, Astrocyte processes flank pyramidal neurons, identified by Nissl substance. C, Astrocyte foot processes abutting blood vessels (bv) are enlarged and depleted of most cytoplasmic organelles. D, E, Toluidine blue-stained 1 μm sections through the CA1 pyramidal cell layer (D) and dentate gyrus (E) demonstrate that pathology is not present in other regions of the hippocampus. Arrows, blood vessels. F, dKO mice fail to preferentially explore a relocated object, indicating impaired spatial memory (p < 0.05 versus mGFAP-Cre mice). Cumulative data from two experiments with 5–6 mice per genotype are shown. G–I, TUNEL assay shows a significant increase in apoptotic cells in dKO brains (p < 0.05, n = 4 brains per genotype). Some apoptotic cells were apparent in close proximity to vacuoles (G). Magnification: (A), 4000×; (B), 4000×; (C), 5000×. Scale bar: (in D) D, E, 10 μm.