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Functional gap junctions in the schwann cell myelin sheath.

Balice-Gordon RJ, Bone LJ, Scherer SS - J. Cell Biol. (1998)

Bottom Line: Gap junctions are localized to periodic interruptions in the compact myelin called Schmidt-Lanterman incisures and to paranodes; these regions contain at least one gap junction protein, connexin32 (Cx32).The radial diffusion of low molecular weight dyes across the myelin sheath was not interrupted in myelinating Schwann cells from cx32- mice, indicating that other connexins participate in forming gap junctions in these cells.Owing to the unique geometry of myelinating Schwann cells, a gap junction-mediated radial pathway may be essential for rapid diffusion between the adaxonal and perinuclear cytoplasm, since this radial pathway is approximately one million times faster than the circumferential pathway.

View Article: PubMed Central - PubMed

Affiliation: Department of Neuroscience, The University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104-6074, USA. rbaliceg@mail.med.upenn.edu

ABSTRACT
The Schwann cell myelin sheath is a multilamellar structure with distinct structural domains in which different proteins are localized. Intracellular dye injection and video microscopy were used to show that functional gap junctions are present within the myelin sheath that allow small molecules to diffuse between the adaxonal and perinuclear Schwann cell cytoplasm. Gap junctions are localized to periodic interruptions in the compact myelin called Schmidt-Lanterman incisures and to paranodes; these regions contain at least one gap junction protein, connexin32 (Cx32). The radial diffusion of low molecular weight dyes across the myelin sheath was not interrupted in myelinating Schwann cells from cx32- mice, indicating that other connexins participate in forming gap junctions in these cells. Owing to the unique geometry of myelinating Schwann cells, a gap junction-mediated radial pathway may be essential for rapid diffusion between the adaxonal and perinuclear cytoplasm, since this radial pathway is approximately one million times faster than the circumferential pathway.

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Pharmacologic blockade of gap junctions with AGA  prevents the diffusion of 5,6-carboxyfluorescein across incisures.  Intensity profiles are illustrated for each panel across a line perpendicular to the long axis of the fiber at the location indicated  by the white arrowhead (scale, 0–255 intensity levels). (A) Filling  of the outer, but not the inner, collar of cytoplasm 1 h after iontophoretic injection of 5,6-carboxyfluorescein after preincubation  in 75 μM AGA. (B) Subsequent confocal analysis of 5,6-carboxyfluorescein in the same fiber shown in A confirmed the absence  of a double train track pattern of staining; shown is a single confocal plane midway through the z projection. (C) Preincubation  of a myelinated fiber in 0.15% DMSO for 1 h did not abolish a  train track pattern of staining After injection of 5,6-carboxyfluorescein. The train track pattern is visible on the right-hand side of  the fiber and also as a doublet in the intensity peak for this region. Bars, 10 μm.
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Figure 7: Pharmacologic blockade of gap junctions with AGA prevents the diffusion of 5,6-carboxyfluorescein across incisures. Intensity profiles are illustrated for each panel across a line perpendicular to the long axis of the fiber at the location indicated by the white arrowhead (scale, 0–255 intensity levels). (A) Filling of the outer, but not the inner, collar of cytoplasm 1 h after iontophoretic injection of 5,6-carboxyfluorescein after preincubation in 75 μM AGA. (B) Subsequent confocal analysis of 5,6-carboxyfluorescein in the same fiber shown in A confirmed the absence of a double train track pattern of staining; shown is a single confocal plane midway through the z projection. (C) Preincubation of a myelinated fiber in 0.15% DMSO for 1 h did not abolish a train track pattern of staining After injection of 5,6-carboxyfluorescein. The train track pattern is visible on the right-hand side of the fiber and also as a doublet in the intensity peak for this region. Bars, 10 μm.

Mentions: Incubation in >1% halothane or >1 μM octanol rapidly induced loss of resting potential, widening of incisures, and disorganization of the Schwann cell membrane (data not shown). Teased fibers maintained in 1–100 μM AGA, however, retained their structural integrity, internalized and then processed calcein AM ester, and excluded ethidium bromide homodimer from the nucleus for more than 4 h. Thus, these concentrations of AGA did not appear to be toxic, and 75 μM AGA was used for subsequent experiments. Nerve fibers were incubated in 75 μM AGA for 40–105 min, then injected iontophoretically with 5,6-carboxyfluorescein and monitored optically for 1–4 h. In pilot experiments, we determined that 30–40 min of incubation in AGA was required to block radial dye transfer in myelinating Schwann cells. The length of time required is probably due to the geometry of the myelin sheath as well as the probable inaccessibility of putative gap junctions to bath applied AGA. In addition to retaining their structural integrity, myelinating Schwann cells incubated in AGA had resting potentials near the normal range (−8.1 ± 1.6 mV, n = 15 fibers from seven mice). Myelinating Schwann cells incubated in AGA did not have a double train track pattern after dye injection (n = six or seven fibers from five mice); the dye remained in the outer collar of cytoplasm (Fig. 7, A and B) and no doublet in the intensity histogram was observed (Fig. 7, A and B, bottom panels). Nerve fibers incubated in 0.15% DMSO (the carrier solution used to dissolve AGA) for a similar length of time also had resting potentials near the normal range (− 9.5 ± 1.1 mV, n = 11 fibers from six mice), but 11 out of 11 fibers showed the double train track pattern after dye diffusion, which was confirmed by the presence of a doublet in the intensity histogram on at least one side of the fiber (Fig. 7 C). Thus, AGA prevented dye from filling the inner collar of Schwann cell cytoplasm, suggesting that functional gap junctions within incisures mediate the diffusion of dye into the inner collar of cytoplasm.


Functional gap junctions in the schwann cell myelin sheath.

Balice-Gordon RJ, Bone LJ, Scherer SS - J. Cell Biol. (1998)

Pharmacologic blockade of gap junctions with AGA  prevents the diffusion of 5,6-carboxyfluorescein across incisures.  Intensity profiles are illustrated for each panel across a line perpendicular to the long axis of the fiber at the location indicated  by the white arrowhead (scale, 0–255 intensity levels). (A) Filling  of the outer, but not the inner, collar of cytoplasm 1 h after iontophoretic injection of 5,6-carboxyfluorescein after preincubation  in 75 μM AGA. (B) Subsequent confocal analysis of 5,6-carboxyfluorescein in the same fiber shown in A confirmed the absence  of a double train track pattern of staining; shown is a single confocal plane midway through the z projection. (C) Preincubation  of a myelinated fiber in 0.15% DMSO for 1 h did not abolish a  train track pattern of staining After injection of 5,6-carboxyfluorescein. The train track pattern is visible on the right-hand side of  the fiber and also as a doublet in the intensity peak for this region. Bars, 10 μm.
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Related In: Results  -  Collection

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Figure 7: Pharmacologic blockade of gap junctions with AGA prevents the diffusion of 5,6-carboxyfluorescein across incisures. Intensity profiles are illustrated for each panel across a line perpendicular to the long axis of the fiber at the location indicated by the white arrowhead (scale, 0–255 intensity levels). (A) Filling of the outer, but not the inner, collar of cytoplasm 1 h after iontophoretic injection of 5,6-carboxyfluorescein after preincubation in 75 μM AGA. (B) Subsequent confocal analysis of 5,6-carboxyfluorescein in the same fiber shown in A confirmed the absence of a double train track pattern of staining; shown is a single confocal plane midway through the z projection. (C) Preincubation of a myelinated fiber in 0.15% DMSO for 1 h did not abolish a train track pattern of staining After injection of 5,6-carboxyfluorescein. The train track pattern is visible on the right-hand side of the fiber and also as a doublet in the intensity peak for this region. Bars, 10 μm.
Mentions: Incubation in >1% halothane or >1 μM octanol rapidly induced loss of resting potential, widening of incisures, and disorganization of the Schwann cell membrane (data not shown). Teased fibers maintained in 1–100 μM AGA, however, retained their structural integrity, internalized and then processed calcein AM ester, and excluded ethidium bromide homodimer from the nucleus for more than 4 h. Thus, these concentrations of AGA did not appear to be toxic, and 75 μM AGA was used for subsequent experiments. Nerve fibers were incubated in 75 μM AGA for 40–105 min, then injected iontophoretically with 5,6-carboxyfluorescein and monitored optically for 1–4 h. In pilot experiments, we determined that 30–40 min of incubation in AGA was required to block radial dye transfer in myelinating Schwann cells. The length of time required is probably due to the geometry of the myelin sheath as well as the probable inaccessibility of putative gap junctions to bath applied AGA. In addition to retaining their structural integrity, myelinating Schwann cells incubated in AGA had resting potentials near the normal range (−8.1 ± 1.6 mV, n = 15 fibers from seven mice). Myelinating Schwann cells incubated in AGA did not have a double train track pattern after dye injection (n = six or seven fibers from five mice); the dye remained in the outer collar of cytoplasm (Fig. 7, A and B) and no doublet in the intensity histogram was observed (Fig. 7, A and B, bottom panels). Nerve fibers incubated in 0.15% DMSO (the carrier solution used to dissolve AGA) for a similar length of time also had resting potentials near the normal range (− 9.5 ± 1.1 mV, n = 11 fibers from six mice), but 11 out of 11 fibers showed the double train track pattern after dye diffusion, which was confirmed by the presence of a doublet in the intensity histogram on at least one side of the fiber (Fig. 7 C). Thus, AGA prevented dye from filling the inner collar of Schwann cell cytoplasm, suggesting that functional gap junctions within incisures mediate the diffusion of dye into the inner collar of cytoplasm.

Bottom Line: Gap junctions are localized to periodic interruptions in the compact myelin called Schmidt-Lanterman incisures and to paranodes; these regions contain at least one gap junction protein, connexin32 (Cx32).The radial diffusion of low molecular weight dyes across the myelin sheath was not interrupted in myelinating Schwann cells from cx32- mice, indicating that other connexins participate in forming gap junctions in these cells.Owing to the unique geometry of myelinating Schwann cells, a gap junction-mediated radial pathway may be essential for rapid diffusion between the adaxonal and perinuclear cytoplasm, since this radial pathway is approximately one million times faster than the circumferential pathway.

View Article: PubMed Central - PubMed

Affiliation: Department of Neuroscience, The University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104-6074, USA. rbaliceg@mail.med.upenn.edu

ABSTRACT
The Schwann cell myelin sheath is a multilamellar structure with distinct structural domains in which different proteins are localized. Intracellular dye injection and video microscopy were used to show that functional gap junctions are present within the myelin sheath that allow small molecules to diffuse between the adaxonal and perinuclear Schwann cell cytoplasm. Gap junctions are localized to periodic interruptions in the compact myelin called Schmidt-Lanterman incisures and to paranodes; these regions contain at least one gap junction protein, connexin32 (Cx32). The radial diffusion of low molecular weight dyes across the myelin sheath was not interrupted in myelinating Schwann cells from cx32- mice, indicating that other connexins participate in forming gap junctions in these cells. Owing to the unique geometry of myelinating Schwann cells, a gap junction-mediated radial pathway may be essential for rapid diffusion between the adaxonal and perinuclear cytoplasm, since this radial pathway is approximately one million times faster than the circumferential pathway.

Show MeSH
Related in: MedlinePlus