Immuno-ultrastructural localization of sodium channels at nodes of Ranvier and perinodal astrocytes in rat optic nerve

Abstract

Immuno-electron microscopic localization of sodium channels at nodes of Ranvier within adult optic nerve was demonstrated with polyclonal antibody 7493. The 7493 antisera, which is directed against purified sodium channels from rat brain, recognizes a 260 kDa protein in immunoblots of the crude glycoprotein fraction from adult rat optic nerve. Intense immunoreactivity with 7493 antisera was observed at nodes of Ranvier. Axon membrane at the node was densely stained, whereas paranodal and internodal axon membrane did not exhibit immunoreactivity. The axoplasm beneath the nodal membrane displayed variable immunostaining. Neither terminal paranodal oligodendroglial loops nor oligodendrocyte plasmalemma were immunoreactive with 7493 antisera. However, perinodal astrocyte processes exhibited intense immunoreactivity with the anti-sodium channel antisera. Optic nerves incubated with pre-immune sera, or with 7493 antisera that had been pre-adsorbed with purified sodium channel protein, displayed no immunoreactivity. These results demonstrate localization of sodium channels at high density at mammalian nodes of Ranvier and in some perinodal astrocyte processes. The latter observation offers support for an active role for perinodal astrocyte processes in the aggregation of sodium channels within the axon membrane at the node of Ranvier.

RETRACTED: Sodium channels in the cytoplasm of Schwann cells.

“…This immunoreactivity of the axon membrane is focal: while the nodal axon membrane is densely stained, adjacent axon membrane beneath the terminal paranodal loops of the Schwann cells and in internodal regions beneath the myelin sheath does not exhibit sodium channel immunoreactivity. This heterogeneous pattern of sodium channel immunoreactivity in the axon membrane is similar to that observed previously in rat optic nerve (14,24) and is expected from the high density of sodium channels previously shown to be present at the node (1-3). As in the rat optic nerve (14), sodium channel immunoreactivity in some fibers is also present in the cytoplasm at the node (but not generally in the internode), and it appears to be associated with some microtubules.…”

“…This heterogeneous pattern of sodium channel immunoreactivity in the axon membrane is similar to that observed previously in rat optic nerve (14,24) and is expected from the high density of sodium channels previously shown to be present at the node (1-3). As in the rat optic nerve (14), sodium channel immunoreactivity in some fibers is also present in the cytoplasm at the node (but not generally in the internode), and it appears to be associated with some microtubules. While it is possible that this represents a fixation artifact due to diffusion of reaction product from a primary binding site at the nodal axon membrane, this observation may well reflect channels being axonally transported, analogous to the recently demonstrated intracellular presence of acetylcholine receptors in cholinoceptive neurons in situ (25,26).…”

“…Sodium channel immunolocalization was carried out as described previously (14). Briefly, rats were anesthetized with sodium pentobarbital (50 mg/kg of body wt) and perfused with 45 paraformaldehyde and 0.01% glutaraldehyde in Sorensen's 0.14 M phosphate buffer.…”

“…Sciatic nerve glycoproteins were prepared essentially as described for optic nerve glycoproteins and for brain and sciatic nerve homogenates (13,14). Adult Sprague-Dawley rats were sacrificed by CO2 inhalation, and sciatic nerves were removed and frozen in liquid nitrogen.…”

Identification of PN1, a predominant voltage-dependent sodium channel expressed principally in peripheral neurons

“…Furthermore, the distribution of PN1 ␣ subunit in cultured DRG cells was similar to that observed in the PC12 cells. To our knowledge, this is the first immunocytochemical demonstration of the targeting of sodium channels to the terminal region, although previous studies have shown that different sodium channel types are targeted to distinct parts of neuronal cell membranes (37)(38)(39)(40)(41)(42). Some staining of PN1 was detected in the cell bodies of both NGF-treated PC12 cells and the cultured DRG neurons.…”