Molecular identity of dendritic voltage-gated sodium channels

Abstract

Active invasion of the dendritic tree by action potentials (APs) generated in the axon is essential for associative synaptic plasticity and neuronal ensemble formation. In cortical pyramidal cells (PCs), this AP back-propagation is supported by dendritic voltage-gated Na+ (Nav) channels, whose molecular identity is unknown. Using a highly sensitive electron microscopic immunogold technique, we revealed the presence of the Nav1.6 subunit in hippocampal CA1 PC proximal and distal dendrites. Here, the subunit density is lower by a factor of 35 to 80 than that found in axon initial segments. A gradual decrease in Nav1.6 density along the proximodistal axis of the dendritic tree was also detected without any labeling in dendritic spines. Our results reveal the characteristic subcellular distribution of the Nav1.6 subunit, identifying this molecule as a key substrate enabling dendritic excitability.

Fig. 1

Somato-dendritic localization of voltage-gated sodium channels. (A) Immunofluorescence localization of the pan-Nav, Nav1.1, Nav1.2 and Nav1.6 subunits in the CA1 area. (B) Double immunofluorescence reaction shows weak pan-Nav immunolabeling along the Kv2.1 subunit immunoreactive somato-dendritic plasma membrane of CA1 PCs. Note the much higher labeling intensity of the AISs. slm: stratum lacunosum-moleculare, sr: stratum radiatum, sp: stratum pyramidale, so: stratum oriens; Scale bars: A, 100 μm; B, 10 μm.

Fig. 2

Immunofluorescence localization of the Nav1.2 and Nav1.6 subunits in CA1 PCs. (A) A double immunofluorescence reaction demonstrates the colocalization of the Nav1.6 and Nav1.2 subunits in the AISs of CA1 PCs. Immunolabeling for the Nav1.2 subunit is confined to the proximal part of the AISs. (B-C) Immunofluorescence double labeling experiments reveal that the majority of the Nav1.2 subunit immunolabeling in stratum radiatum (sr) co-localizes with the presynaptic marker vGluT1 (B), but does not co-localize with the Kv4.2 subunit (C), a K⁺ channel subunit that is known to be present in PC dendritic shafts and spines. (D-E) An immunofluorescence reaction for the Nav1.6 subunit in the strata pyramidale (sp) and proximal radiatum (sr) of the CA1 area demonstrates weak plasma membrane-like labeling of PC proximal apical dendrites. Note the much higher labeling intensity of the AISs. (F) A double immunofluorescence reaction in the stratum radiatum shows the colocalization of pan-Nav (red) and the Nav1.6 subunit (green) in a strongly immunopositive AIS and its parent apical dendrite. Scale bars: A, E, F, 5 μm; B-D, 10 μm.

Fig. 3

High resolution immunogold localization of the Nav1.6 subunit in AISs and nodes of Ranvier of CA1 PCs. (A) A high density of gold particles labeling the Nav1.6 subunit is found on the P-face of an AIS in the stratum pyramidale. Note that the immunogold particles avoided the area of the postsynaptic density (PSD) of an axo-axonic synapse. (B) An AIS is co-labeled for the Nav1.6 subunit (15 nm gold) and the AIS marker pan-Neurofascin (10 nm gold). (C) A high magnification view of the boxed area shown in B. (D) High magnification image of the P-face of an AIS labeled for the Nav1.6 subunit (15 nm gold) and Ankyrin-G (10 nm gold). (E) A low magnification image shows a node of Ranvier (R) of a myelinated axon (m) in the alveus. (F) A high magnification view of the node of Ranvier shown in E. The P-face of the node of Ranvier membrane contains a high density of gold particles labeling the Nav1.6 subunit. Note the lack of labeling over the myelin (E and F). (G) A gradual increase in the intensity of Nav1.6 immunofluorescence is found along the proximo-distal axis of CA1 PC AISs. (H) A low magnification image of a replica shows a fragment of a somatic plasma membrane and an emerging AIS (red). (I-L) High magnification images of the boxed areas shown in panel H. Images taken of the soma (L) and of the AIS at various distances from the soma (I-K) demonstrate an increase in the density of gold particles labeling the Nav1.6 subunit along the proximo-distal axis of the AIS (I). Scale bars: A, B, F, 200 nm; C, D, I-L, 100 nm; E, 500 nm; G, 5 μm; H, 1 μm.

Fig. 4

Somato-dendritic localization of the Nav1.6 subunit in CA1 PCs using SDS-FRL. (A) Low density of gold particles labeling the Nav1.6 subunit is found on P-face of the somatic plasma membrane of a CA1 PC. No immunolabeling is seen on the E-face of a neighboring PC soma. (B) Low magnification image of the P-face of a thick, spiny apical dendrite (d) of a CA1 PC in the proximal stratum radiatum labeled for the Nav1.6 subunit. Bar graphs show the densities of gold particles (mean ± SD) in somato-dendritic subcellular compartments of CA1 PCs, some of which differ significantly from the nonspecific labeling (ANOVA: p < 0.001; Dunnett’s post hoc test: soma, prox. apic. dendr., prox. obl. dendr., dist. apic. dendr.: p < 0.05; n = 5 rats). (C) A high magnification image of the boxed area shown in B. Gold particles labeling the Nav1.6 subunit are distributed on the dendritic shaft (d), but avoid the dendritic spine (sp). (D) The P-face of a spiny oblique dendrite (Obl. d) in the proximal stratum radiatum contains a low density of gold particles for the Nav1.6 subunit (arrows), with no particle in the spine (sp). Scale bars: B, 1 μm; A, C, D, 200 nm.