Genetic and physiological evidence that oligodendrocyte gap junctions contribute to spatial buffering of potassium released during neuronal activity

Abstract

Mice lacking the K+ channel Kir4.1 or both connexin32 (Cx32) and Cx47 exhibit myelin-associated vacuoles, raising the possibility that oligodendrocytes, and the connexins they express, contribute to recycling the K+ evolved during neuronal activity. To study this possibility, we first examined the effect of neuronal activity on the appearance of vacuoles in mice lacking both Cx32 and Cx47. The size and number of myelin vacuoles was dramatically increased when axonal activity was increased, by either a natural stimulus (eye opening) or pharmacological treatment. Conversely, myelin vacuoles were dramatically reduced when axonal activity was suppressed. Second, we used genetic complementation to test for a relationship between the function of Kir4.1 and oligodendrocyte connexins. In a Cx32-null background, haploinsufficiency of either Cx47 or Kir4.1 did not affect myelin, but double heterozygotes developed vacuoles, consistent with the idea that oligodendrocyte connexins and Kir4.1 function in a common pathway. Together, these results implicate oligodendrocytes and their connexins as having critical roles in the buffering of K+ released during neuronal activity.

Figure 1.

Massive vacuolation in the Cx32/Cx47 dKO optic nerves coincides with eye-opening. A, Toluidine blue-stained semithin sections of optic nerves from dKO mice and their WT littermates at the indicated ages. Scale bar, 100 μm. B, Quantification of total vacuolated area (μm²/0.1 mm²) in dKO optic nerves. Error bars indicate SEM.

Figure 2.

Vacuolation in Cx32/Cx47 dKO optic nerves is reduced when axonal activity is suppressed. Intraocular injections of TTX were performed at P11 and P13; optic nerves and primary visual cortex were harvested at P15. A, Semithin sections from the optic nerve associated with an injected eye reveal a dramatic reduction in vacuolation compared with the contralateral control nerve. Scale bars: top row, 100 μm; bottom row, 10 μm. B, Quantification of small (<20 μm²) and large (>20 μm²) vacuoles, as well as the total vacuolated area (μm²/0.1 mm²) and the number of myelinated axons (per 0.1 mm²) in TTX-injected (+) and -uninjected/contralateral (−) optic nerves. The BDNF mRNA levels were normalized to HPRT mRNA and are presented as a ratio of contralateral injected visual cortex/contralateral uninjected visual cortex. Error bars indicate SEM. TTX injections result in fewer vacuoles, a reduction in total vacuolated area, and lower BDNF mRNA ratios.

Figure 3.

Vacuolation in Cx32/Cx47 dKO optic nerves is increased when axonal activity is stimulated. Intraocular injections of cholera toxin were performed at P9; optic nerves and visual cortex were harvested at P11. A, Optic nerves associated with the injected eye show a dramatic increase in vacuolation. Scale bars: top row, 100 μm; bottom row, 10 μm. B, Quantification of small (<20 μm²) and large (>20 μm²) vacuoles, total vacuolated area (μm²/0.1 mm²), and the number of myelinated axons per 0.1 mm² optic nerve, from cholera toxin-injected (+) and contralateral (−) eyes, as well as the BDNF mRNA ratio. Cholera toxin injections result in more vacuoles, an increase in vacuolated area, and higher BDNF mRNA levels.

Figure 4.

Pathological findings in Kir4.1-null mice. These are images of transverse sections of P10 spinal cords from Kir4.1-null mice or their WT littermates. Semithin sections show abundant vacuoles (v) in the white matter of Kir4.1-null (B) but not WT (A) spinal cords, in which a capillary (c) is indicated. C–F, Electron micrographs of Kir4.1-null white matter. C, D, F, Typical vacuoles that are associated with sheaths (arrowheads). E, An atypical vacuole that separates an axon (ax) from its myelin sheath. D, Part of a normal appearing oligodendrocyte (n). F, Part of an apoptotic oligodendrocyte (a). Scale bars: A, B, 10 μm, C–F, 1 μm.

Figure 5.

Genetic evidence that Kir4.1 channels and gap junctions function in a common pathway: pathological findings in Cx32^−/Y, Cx47^+/−, Kir4.1^+/− spinal cords. Low-magnification images of toluidene blue-stained semithin transverse sections of spinal cord gray matter reveal the extent of vacuolation in Cx32^−/Y, Cx47^+/−, Kir4.1^+/− mice (C) but not in controls (A, B). D–G, These are images of transverse sections of spinal cord gray matter from 5-week-old mice. Capillaries (c) but not vacuoles (v) are present in this field from a Cx32^+/−, Cx47^+/−, Kir4.1^+/− (D), but vacuoles are abundant in Cx32^−/Y, Cx47^+/−, Kir4.1^+/− (E–G) spinal cords. Electron microscopy shows a typical vacuole that is partly surrounded by a myelin sheath (F), as well as electron-dense cellular processes (G; asterisks), which are also a feature of this genotype. Scale bars: A, B, 10 μm; C, D, 1 μm.

Figure 6.

Possible routes for the dispersal of K⁺ released during neuronal activity. Neuronal Kv1.1 and Kv1.2 channels are localized to the junxtaparanodal axolemma (1). Thus, K⁺ ions released through these channels during neuronal activity enter a private extracellular compartment bounded by axoglial junctions at the paranodes. Some K⁺ ions could leak through these junctions, gaining direct access to astrocytes (5) for uptake, but Na-K-ATPases in axolemma (2) and oligodendrocyte plasma membrane (3) likely account for the majority of uptake. Once in the periaxonal cytoplasm of the oligodendrocyte, K⁺ could migrate though reflexive gap junctions located in the paranodal membranes (4) to reach the outer layer of myelin and the oligodendrocyte cell body. Then, K⁺ could gain entry to the astrocyte cytoplasm via heterotypic gap junctions between oligodendrocyte and astrocyte (6). Finally, Kir4.1 channels located in astrocyte end feet at capillaries and the glia limitans (8) would contribute to “K⁺ siphoning” and removal.