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Molecular Identity of Dendritic Voltage-Gated Sodium Channels

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Science  14 May 2010:
Vol. 328, Issue 5980, pp. 906-909
DOI: 10.1126/science.1187958

Counting Na+ Channels One by One

Understanding how nerve cells integrate their synaptic inputs and generate their output signals requires the identification of voltage-dependent ion channels on the axo-somato-dendritic surface of central neurons. Using improved ultrastructural immunocytochemistry techniques, Lorincz and Nusser (p. 906) found that a newly described voltage-gated sodium channel, Nav1.6, was present not only at nodes of Ranvier and axon initial segments but also at much lower, but functionally significant levels, in dendrites of CA1 pyramidal cells. However, other brain Na+ channels were not present in these dendrites, suggesting that dendritic sodium spikes result from somatic activation of this particular type of sodium channel.

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.

Associative synaptic plasticity in cortical pyramidal cells (PC) is the most widely accepted cellular model of learning. An essential prerequisite of the model is that input synapses, which are distributed over an enormously large dendritic tree, must be capable of sensing the precise timing of the output signal. The most likely mechanism for this is the active invasion of the dendritic tree by fast sodium action potentials (APs) initiated in the axon initial segment (AIS). Modification of back-propagating APs therefore holds tremendous potential for altering synaptic plasticity and forming of neuronal representations. Voltage-gated Na+ (Nav) currents have been detected in hippocampal and neocortical PC dendrites, where they not only support AP back-propagation but also underlie nonlinear synaptic integration and dendritic sodium spike generation [(16) and reviewed in (710)]. Patch-clamp experiments demonstrate that axonal and somatodendritic Nav currents differ in their activation and inactivation properties (11, 12), indicating either different Nav subunit compositions or distinct posttranslational modifications of identical subunits (1214). However, the lack of subunit-specific drugs precludes the unequivocal identification of the subunit composition of the axosomatodendritic Nav channels using functional approaches. Immunohistochemistry with subunit-specific antibodies offers an alternative experimental approach to address this issue. Nav1.1, Nav1.2, and Nav1.6 are the Nav subunits expressed in adult brains (15, 16). They have been detected in nodes of Ranvier, AISs, and preterminal axons using immunofluorescence localization (1722). Unfortunately, this method has so far failed to visualize any of these Nav subunits in somatodendritic compartments, most likely due to its insufficient sensitivity. We modified the immunofluorescence technique to increase its sensitivity and employed a highly sensitive, quantitative electron microscopic (EM) immunogold method [SDS-digested freeze-fracture replica-labeling (SDS-FRL)] (2325) to identify the Nav subunit content of the somatodendritic plasma membranes of CA1 PCs.

In conventional immunofluorescence localization techniques, the tissue is usually fixed with a phosphate-buffered fixative containing paraformaldehyde, resulting in a fixation that masks antigens in many subcellular compartments, including, for example, postsynaptic densities and AISs (26). The visualization of antigens in some of these compartments might be possible after antigen retrieval, such as microwave irradiation or pepsin digestion (27, 28). We used a low-pH–based fixation approach (see supporting online material) that vastly increased the strength of the reactions for almost all γ-aminobutyric acid type A (GABAA) and glutamate receptors, and voltage-gated K+ and Na+ channels, and allowed the visualization of these proteins in postsynaptic membranes and AISs without antigen retrieval. Using this approach, we first employed pan-Nav antibodies to visualize all Nav subunits in the hippocampal CA1 area of the adult rat. Immunoreactions with pan-Nav antibodies revealed a very strong labeling of AISs and nodes of Ranvier in stratum pyramidale (SP) overlying a rather homogeneous staining of the strata oriens (SO), radiatum (SR) and lacunosum-moleculare (SLM) (Fig. 1A). The increased sensitivity afforded by the low-pH fixation condition allowed the visualization of pan-Nav immunosignal associated with somatic and proximal dendritic plasma membranes of PCs (Fig. 1B) that were also outlined by strong Kv2.1 subunit immunoreactivity. The intensity of the dendritic pan-Nav labeling was much weaker than that of the AISs, consistent with functional predictions (29). Interestingly, in some PCs, strongly pan-Nav–labeled AISs originated from the apical dendrites and a stronger pan-Nav labeling was associated with the plasma membrane of these dendrites around the origin of the AIS (Fig. 2F and fig. S1). Our pan-Nav immunofluorescence reactions visualized Nav channels in the somatodendritic compartments of PCs and revealed large differences in Nav density between AISs and somatodendritic plasma membranes. However, the subunit composition, the presence or absence of Nav channels in small dendritic compartments such as small oblique dendrites and dendritic spines, and the exact quantitative difference in Nav density between distinct subcellular compartments remained elusive.

Fig. 1

Somatodendritic 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 somatodendritic 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), 50 μm; (B), 10 μm.

To address the first of these issues, we carried out immunofluorescence reactions with specific antibodies against the Nav1.1, Nav1.2, and Nav1.6 subunits. After confirming the specificity of the immunoreactions (figs. S2 and S3A), we investigated their distributions in the CA1 area. The labeling pattern obtained with the pan-Nav antibodies was essentially the sum of the patterns obtained for these three subunits (Fig. 1A). Immunosignal for the Nav1.1 subunit was restricted to small axonlike profiles in the SP; diffuse signal for the Nav1.2 subunit was observed in the neuropil of the SO, SR, and SLM; and intense Nav1.6 labeling was confined to AISs and nodes of Ranvier (Fig. 1A). Double immunolabeling for Kv3.1b and Nav1.1 subunits demonstrated that this Nav subunit is present in small axonlike processes and AISs of Kv3.1b immunopositive GABAergic interneurons (fig. S3), but not in PCs, in agreement with a previous study (19). Double immunofluorescence reactions revealed the presence of the Nav1.2 subunit in the proximal part of PC AISs (Fig. 2A) (17), but no Nav1.2 subunit immunoreactivity could be detected in the plasma membranes of somata and proximal apical dendrites. The diffuse Nav1.2 subunit immunolabeling of the neuropil was associated with vGluT1 immunopositive presynaptic axons (Fig. 2B) but not with Kv4.2 immunoreactive dendritic shafts or spines of CA1 PCs (Fig. 2C), demonstrating the predominantly presynaptic expression of this subunit. Very intensely labeled AISs and nodes of Ranvier dominated the pictures obtained for the Nav1.6 subunit. In addition, in our best reactions, very weak plasma membrane–like labeling of PC apical dendrites could be observed (Fig. 2, D and E). This labeling decreased in intensity when individual dendrites were followed from their somata toward their apical tips (Fig. 1A). Weak dendritic Nav1.6 labeling could be detected in those PCs in which AISs originated from the apical dendrites (Fig. 2F). However, even in our best reactions, small-diameter oblique apical and basal dendrites, as well as dendritic spines, remained immunonegative. These results provide molecular identification of dendritic Nav channels and predict complex subcellular compartment- and distance-dependent regulation of the cell-surface density of Nav1.6 in PC dendritic trees.

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 and C) Immunofluorescence double-labeling experiments reveal that the majority of the Nav1.2 subunit immunolabeling in stratum radiatum (sr) colocalizes with the presynaptic marker vGluT1 (B) but does not colocalize with the Kv4.2 subunit (C), a K+ channel subunit that is known to be present in PC dendritic shafts and spines. (D and 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), and (F), 5 μm; (B) to (D), 10 μm.

To identify the potential presence of the Nav1.6 subunit in small dendritic compartments and to quantify the differences in the plasma membrane density of Nav1.6 in distinct subcellular compartments, we adopted SDS-FRL (23, 24), a recently developed quantitative EM immunolocalization method with exceptionally high sensitivity. The labeling efficiency for synaptic AMPA receptors of ~100% using SDS-FRL (30) is about 2.5 times as high as that achieved with a postembedding immunogold localization of the same subunits with an identical antibody (31). A labeling efficiency of 100% means that, on average, every protein is represented by a gold particle with a localization method that has a spatial resolution of ~25 nm. We therefore exploited the potential of this method for Nav1.6 localization by preparing carbon-platinum-carbon replicas of the CA1 area after high-pressure freezing and freeze-fracturing, and immunoreacted them with our antibodies to Nav1.6 (18). First we sought to examine the immunogold content of AISs (Fig. 3), the most intensely labeled subcellular structures found in the immunofluorescence reactions. Many elongated protoplasmic face (P-face) membrane structures in SP and SO contained a very high density of gold particles (Fig. 3A). These structures were then molecularly identified as AISs in double-labeling experiments with AIS markers such as pan-Neurofascin and Ankyrin-G (Fig. 3, B to D). The labeling on the P-face membranes is consistent with the cytoplasmic location of the epitope recognized by our antibody (intracellular loop between domains II and III), given the accepted transmembrane topology of the Nav1.6 subunit. In a few cases, AISs were fractured together with a fragment of the corresponding somatic plasma membrane (Fig. 3H), allowing us to assess how Nav1.6 density changes in the AIS as a function of distance from the soma. The graded increase in the density of the Nav1.6 subunit along the proximodistal axis of PC AISs observed in the immunofluorescence reactions (Fig. 3G) could also be captured in replicas (Fig. 3, H to L). Furthermore, gold particles labeling the Nav1.6 subunit on AISs consistently avoided the perisynaptic and synaptic membrane areas of axo-axonic GABAergic synapses, identified by clusters of intramembrane particles (Fig. 3A and fig. S4B) or by intense clustering of gold particles labeling neuroligin-2 (fig. S4C) or GABAA receptor β3 subunit (fig. S4, D and E). The absence of the Nav1.6 subunit in GABAergic axo-axonic synapses is another feature that is reliably captured with SDS-FRL, as well as with double immunofluorescence labeling for Nav1.6 and, for example, the GABAA receptor α2 subunit (fig. S4A). Large somatic P-face membrane structures in SP contained many more gold particles for the Nav1.6 subunit than membranes fractured to the extracellular face (E-face) (Fig. 4A). A similar picture was observed on proximal apical dendrites, where P-face dendritic shaft membranes contained gold particles (Fig. 4, B and C), consistent with our fluorescent observations. After the successful visualization of high-density Nav1.6 labeling in AISs and a much lower density in somata and proximal apical dendrites, we turned our attention to small dendritic compartments, which remained immunonegative even in our best fluorescence reactions. Dendritic shafts and spines of apical oblique dendrites were sampled at proximal (25 to 50 μm from SP) and distal (100 to 150 μm) locations in the SR, as well as dendritic shafts in SLM. Gold particles were observed on the P-face of dendritic shaft membranes in all locations, but dendritic spines were consistently immunonegative (Fig. 4, C and D). An intrinsic property of immunogold localization is that gold particles can be found in any location by chance. Therefore, the presence of Nav1.6 subunits cannot be concluded just from the presence of a few gold particles in a given compartment but requires a quantitative assessment of whether the particle density in a given location is significantly higher than that expected from a random process. We determined the nonspecific background labeling within our reactions by counting gold particles on the E-face plasma membranes (where the intracellular epitope of Nav subunits is not present) and found that on average 1.2 ± 0.4 gold particles (n = 5 rats) are expected to be present in every μm2 membrane by chance (fig. S5). We then determined the density of gold particles labeling the Nav1.6 subunit on P-face plasma membranes of somata [6.3 ± 1.8 gold/μm2 (mean ± SD); n = 5 rats], proximal apical dendrites (6.5 ± 1.5 gold/μm2), proximal oblique dendrites (4.6 ± 1.4 gold/μm2), distal apical dendrites (3.5 ± 1.4 gold/μm2), distal oblique dendrites (3.0 ± 1.7 gold/μm2), and dendrites in SLM (2.1 ± 0.6 gold/μm2). Of these, somata, proximal apical, proximal oblique, and distal apical dendrites displayed a significantly higher density of gold particles than the background labeling (Fig. 4B) [one-way analysis of variance (ANOVA) with Dunnett’s post-hoc test, P < 0.05]. Thus, the exceptionally high sensitivity of the SDS-FRL method allowed us to reveal low, but significant, densities of the Nav1.6 subunit in these dendritic compartments, which remained immunonegative with the immunofluorescence method. Along these lines, it should be noted that the presence of low densities of other Nav subunits (e.g., Nav1.2) in somatodendritic compartments cannot be excluded based on immunofluorescence data only, but highly sensitive reactions must be accomplished with SDS-FRL to unequivocally determine their presence or absence in identified subcellular compartments. Finally, we compared the Nav1.6 subunit density in somatodendritic compartments to that found in AISs and nodes of Ranvier. Nodes of Ranvier in the alveus were very strongly immunolabeled for the Nav1.6 subunit (Fig. 3, E and F), containing immunogold particles at almost twice the density of that observed in AISs (346.3 ± 82.9 versus 186.9 ± 29.9 gold/μm2). The AISs contained densities of gold particles that were 39 ± 9 and 36 ± 6 times as high as those in somata and proximal apical dendrites, respectively. These ratios are very similar to those estimated (29) for neocortical layer 5 PCs using whole-cell Nav current recordings (a factor of ~45) and Na+ imaging (a factor of ~30). When dendritic compartments at the same distance from the soma were compared, we found that oblique dendrites contain less Nav1.6 subunit than main apical dendrites. There was also a clear tendency for a distance-dependent reduction in the density of gold particles along the proximodistal axis of the dendritic tree (Fig. 4B). We could not detect immunogold particles labeling the Nav1.6 subunit above the background level in preterminal axons, axon terminals, or dendritic spines.

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. The immunogold particles avoided the area of the postsynaptic density (PSD) of an axo-axonic synapse. (B) An AIS is colabeled 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 proximodistal 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 to L) High-magnification images of the boxed areas shown in (H). Images taken of the soma (L) and of the AIS at various distances from the soma [(I) to (K)] demonstrate an increase in the density of gold particles labeling the Nav1.6 subunit along the proximodistal axis of the AIS (I). Scale bars: (A), (B), and (F), 200 nm; (C), (D), and (I) to (L), 100 nm; (E), 500 nm; (G), 5 μm; (H), 1 μm.

Fig. 4

Somatodendritic 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 the 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 somatodendritic subcellular compartments of CA1 PCs, some of which differ significantly from the nonspecific labeling (ANOVA: P < 0.001; Dunnett’s post hoc test: soma, proximal apical dendrites, proximal oblique dendrites, distal apical dendrites; 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), and (D), 200 nm.

Using an improved immunofluorescence method and SDS-FRL, we identify Nav1.6 as the prominent somatodendritic Nav channel subunit in hippocampal PCs. Using SDS-FRL, we demonstrate a characteristic subcellular distribution pattern of the Nav1.6 subunit on the axosomatodendritic compartments of PCs, revealing a very dramatic drop (by a factor of 40 to 70) in density from nodes of Ranvier and AISs to somata, and a distance-dependent decrease in density along the proximodistal axis of the dendritic tree. By exploiting the exquisite sensitivity of the SDS-FRL method, we obtained a gold particle density for the Nav1.6 subunit of 187 ± 30 (gold/μm2) in AISs of CA1 PCs. A previous study (29) using in vitro electrophysiology, Na+ imaging, and multicompartmental modeling estimated a conductance density of Nav channels in neocortical layer 5 PC AISs of 2500 pS/μm2; given a 17-pS single-channel conductance, this predicts a functional channel density of ~150 channels per μm2. If CA1 PCs have an AIS Nav channel density similar to layer 5 PCs, we can conclude that the labeling efficiency of our SDS-FRL method is close to 100%, similar to that obtained for synaptic AMPA receptors (30). Given this estimated labeling efficiency, our results predict a Nav channel density (channels per μm2 plasma membrane) of 5 in somata and proximal apical dendrites, 3 in proximal oblique dendrites, and 2 in distal apical dendrites. The differences in the density of Nav1.6 in somatic and dendritic compartments represent only one level of complexity. Additional compartment-dependent variations in, for example, the phosphorylation states of the channels (12) could further increase functional diversity. Our results demonstrate that Nav1.6 is the main Nav subunit in the somatodendritic compartments of CA1 PCs, whereas both Nav1.6 and Nav1.2 subunits are present in AISs (17). It remains to be determined whether the axosomatic difference in the subunit composition of Nav channels is solely responsible for the reported differences in activation threshold or whether additional differential regulation of axonal versus somatic Nav channels also exists. Irrespective of that, our data support the view that the large axonal Nav channel density is primarily responsible for the fact that the axon has the lowest voltage threshold for generating action potentials (32).

Supporting Online Material

www.sciencemag.org/cgi/content/full/328/5980/906/DC1

Materials and Methods

Figs. S1 to S5

References and Notes

  1. A.L. is the recipient of a János Bolyai Scholarship of the Hungarian Academy of Sciences. Z.N. is the recipient of a European Young Investigator Award (www.esf.org/euryi) and Wellcome Trust Project and Equipment grants. We thank D. Rónaszéki and É. Dobai for their excellent technical help, W. Sieghart and J.-M. Fritschy for antibodies to the GABAA receptors, and M. Eyre and M. Farrant for their comments on the manuscript.
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