Paranodal interactions regulate expression of sodium channel subtypes and provide a diffusion barrier for the node of Ranvier

Abstract

The node of Ranvier is a distinct domain of myelinated axons that is highly enriched in sodium channels and is critical for impulse propagation. During development, the channel subtypes expressed at the node undergo a transition from Nav1.2 to Nav1.6. Specialized junctions that form between the paranodal glial membranes and axon flank the nodes and are candidates to regulate their maturation and delineate their boundaries. To investigate these roles, we characterized node development in mice deficient in contactin-associated protein (Caspr), an integral junctional component. Paranodes in these mice lack transverse bands, a hallmark of the mature junction, and exhibit progressive disruption of axon-paranodal loop interactions in the CNS. Caspr mutant mice display significant abnormalities at central nodes; components of the nodes progressively disperse along axons, and many nodes fail to mature properly, persistently expressing Nav1.2 rather than Nav1.6. In contrast, PNS nodes are only modestly longer and, although maturation is delayed, eventually all express Nav1.6. Potassium channels are aberrantly clustered in the paranodes; these clusters are lost over time in the CNS, whereas they persist in the PNS. These findings indicate that interactions of the paranodal loops with the axon promote the transition in sodium channel subtypes at CNS nodes and provide a lateral diffusion barrier that, even in the absence of transverse bands, maintains a high concentration of components at the node and the integrity of voltage-gated channel domains.

Figure 1.

Nodes of Ranvier in the PNS and CNS are longer in Caspr mutant mice. Sciatic nerves from 4-month-old wild-type (a) and Caspr mutant (b) mice were stained for NrCAM (red) and neurofilament (green); insets show NrCAM and neurofilament immunolabeling of single nodes labeled with dashed boxes. Optic nerves from wild-type (c) and Caspr mutant (d) littermates were stained for βIV spectrin (red) and neurofilament (green). The nodes in the wild type are punctate, whereas nodes in the mutant are more variable in length and shape. Nodal lengths were quantitated in sciatic nerves (e) and optic nerves (f). The total number of nodes were binned into 0.50 μm increments and graphed for both wild-type (white) and Caspr (gray) mutants; Caspr-deficient nerves exhibit a greater range in nodal length in both the PNS and CNS. Scale bars, 10 μm.

Figure 2.

Nodes in the CNS of Caspr mutants increase in length with age. Optic nerve sections from age-matched wild-type and Caspr knock-out mice at 3 weeks, 6 months, and 2 years of age stained forβIV spectrin are shown (a-f). Scale bar, 10 μm. Node lengths were quantitated (g) and are presented as means ± SEM. In the sciatic nerve (SN), the length of the node in both wild-type and Caspr mutant mice increases slightly with age. In the optic nerve (ON), the length of the node decreases slightly in the wild-type mice but increases substantially with age in the Caspr mutant. Nodes are significantly longer in the Caspr mutants than age-matched wild-type nerves in both the CNS and PNS at all ages (^*p < 0.0001).

Figure 3.

Concentration of nodal components is related to node length. The intensities of fluorescence staining for βIV spectrin (a,b) and Na⁺ channel (c,d) were measured along the length of optic nerve nodes in 4-month-old mice. Nodes are binned based on their length into 0.5 μm increments; each bin is plotted as a different color (see key). Nodes in the wild type (a,c) are of uniform size and have similar intensities. Caspr knock-outs (b,d) have many more bins because of greater variation in nodal length. The intensity of βIV spectrin (b) and Na⁺ channel (d) staining decreases as a function of length.

Figure 4.

Ultrastructure and freeze fracture of the paranodal region in Caspr mutants. Electron micrographs of paranodal regions from optic nerves of 2-year-old wild-type (a) and Caspr mutant (b) mice. In the wild-type optic nerve, paranodal loops are regularly arranged and closely apposed to the axon. In contrast, in this example from the Caspr mutant optic nerve (b), a single myelin sheath separates into three distinct sets of myelin lamellae (numbered in the figure), each of which terminate with both inverted and everted loops. The outermost set of lamellae (3) has retracted from the node, which is just visible at the bottom right. A myelin sheath (^*) from another cell overrides this paranodal region. c, Freeze-fracture replica showing a palisade of presumptive paranodal processes labeled above (^*). Two of these display macular particle patches resembling the gap junctions (GJ) characteristic of astrocyte processes. A thin cellular process (black arrow) intervenes between these and an E-face (AE) containing node-like membrane particles in a density of ∼800/μm² that spread well into the paranodal region. The proportion of particles 10 nm or greater is 56%, consistent with the proportion in the normal nodal axolemma. Scale bars: a-c, 0.5 μm. d,e, Details of gap junction-like patches in freeze-fracture replicas of membranes within paranodal processes. d, E-face pits; e, P-face particles. Scale bar, 0.1 μm.

Figure 5.

Na_v1.2 and Na_v1.6 expression in optic nerves from wild-type and mutant mice. Optic nerve sections from 2-year-old wild-type (+/+) and Caspr mutant (-/-) littermates were stained with anti-Na_v1.2 (green), anti-βIV spectrin (red), and anti-Na_v 1.6 (blue) antibodies. Na_v 1.2-positive nodes are not present in the wildtype (a,c), whereas the Caspr knock-out continues to express significant numbers of Nav 1.2-positive nodes (appear as yellow nodes in the merged images in d,f). Na_v 1.6 is expressed at almost all the nodes of Ranvier in the wild type (magenta in the merged images in b,c) but at very few of the Caspr knock-out nodes (e,f). A few nodes (red in all panels) do not express either isoform and are indicated with arrowheads. Scale bar, 10 μm.

Figure 6.

Expression of Na_v1.2 and Na_v1.6 in wild-type and Caspr mutant mice of different ages. The percentage of nodes that are Na_v1.2 or Na_v1.6 positive in wild-type (+/+) or Caspr null (-/-) mice was quantitated at 14 d, 21 d, 4-6 months, and at 2 years. In the wild-type nerves, Na_v1.2 is expressed in the majority of nodes but is substantially replaced by Na_v1.6 at 4-6 months. In the Caspr mutants, Na_v1.2 is expressed in approximately half of the nodes at all time points, and the proportion of Na_v1.6-positive nodes does not increase further after P21.

Figure 7.

The expression of Na_v1.2 and Na_v1.6 in P10 sciatic nerves. Wild-type (+/+) sciatic nerves from P10 mice were stained with anti-Caspr antibodies (green) and anti-Na_v1.2 (red) (a-c) or anti-Na_v1.6 antibodies (red) (panels g-i). Caspr mutant (-/-) sciatic nerves were labeled with anti-Na⁺ channel antibodies (green) and anti-Na_v1.2 (red) (d-f) or anti-Na_v1.6 (j-l) antibodies. Very few nodes in the wild type at this age are Na_v1.2 positive (one is indicated with an arrow in a,c); in contrast, many of the Caspr mutant nodes are Na_v1.2 positive (d,f). All nodes were Na_v 1.6 positive in the wild-type and knock-out mice, indicating that Na_v1.2 and Na_v1.6 are frequently coexpressed in the P10 knock-out nerves. Scale bar, 20 μm.

Figure 8.

Potassium channel distribution is aberrant in Caspr-deficient mice. Optic nerve sections of 4-month-old (a,b) and 2-year-old (c,d) optic nerves from wild-type (+/+) and Caspr mutants (-/-) were triple stained for K_v1.1 (red), Caspr (blue), andβIV spectrin (green). K_v1.1 is present in many of the juxtaparanodes of the wild type (a,c) but only a few of the paranodes of the Caspr mutants (b,d), particularly at 2 years. K_v1.1 expressed in the paranodes of the mutants frequently flanks well delineated nodes; two examples are indicated (d; yellow arrowheads). Sciatic nerves from wild-type (e,g) and Caspr mutant (f,h) littermates were triple stained for K_v1.1 (red),βIV spectrin (green), and MBP (blue) (e,f) or Kv1.1 (red) alone (g,h). K_v1.1 is expressed in the juxtaparanodes of wild-type mice (e) and the paranodes of Caspr mutants (f). Deficient K_v 1.1 expression in the paranodes of Caspr mutants is associated with attenuatedβIV spectrin at the node (f, white arrow). There are more Schmidt-Lanterman clefts in the Caspr mutant (h, white arrowheads) than in the wild type (g, white arrowheads). K_v1.1 is also occasionally expressed in a band along the axon (h, asterisk). Scale bars, 10 μm.