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Characterization of the axon initial segment (AIS) of motor neurons and identification of a para-AIS and a juxtapara-AIS, organized by protein 4.1B.

Duflocq A, Chareyre F, Giovannini M, Couraud F, Davenne M - BMC Biol. (2011)

Bottom Line: We also identified in all α motor neurons a hemi-node-type organization, with a contactin-associated protein (Caspr)+ paranode-type, as well as a Caspr2+ and Kv1+ juxtaparanode-type compartment, referred to as a para-AIS and a juxtapara (JXP)-AIS, adjacent to the AIS, where the myelin sheath begins.We found that Kv1 channels appear in the AIS, para-AIS and JXP-AIS concomitantly with myelination and are progressively excluded from the para-AIS.Protein 4.1B plays a key role in ensuring the proper molecular compartmentalization of this hemi-node-type region.

View Article: PubMed Central - HTML - PubMed

Affiliation: INSERM UMRS 952, 9 Quai St Bernard, F-75005, Paris, France.

ABSTRACT

Background: The axon initial segment (AIS) plays a crucial role: it is the site where neurons initiate their electrical outputs. Its composition in terms of voltage-gated sodium (Nav) and voltage-gated potassium (Kv) channels, as well as its length and localization determine the neuron's spiking properties. Some neurons are able to modulate their AIS length or distance from the soma in order to adapt their excitability properties to their activity level. It is therefore crucial to characterize all these parameters and determine where the myelin sheath begins in order to assess a neuron's excitability properties and ability to display such plasticity mechanisms. If the myelin sheath starts immediately after the AIS, another question then arises as to how would the axon be organized at its first myelin attachment site; since AISs are different from nodes of Ranvier, would this particular axonal region resemble a hemi-node of Ranvier?

Results: We have characterized the AIS of mouse somatic motor neurons. In addition to constant determinants of excitability properties, we found heterogeneities, in terms of AIS localization and Nav composition. We also identified in all α motor neurons a hemi-node-type organization, with a contactin-associated protein (Caspr)+ paranode-type, as well as a Caspr2+ and Kv1+ juxtaparanode-type compartment, referred to as a para-AIS and a juxtapara (JXP)-AIS, adjacent to the AIS, where the myelin sheath begins. We found that Kv1 channels appear in the AIS, para-AIS and JXP-AIS concomitantly with myelination and are progressively excluded from the para-AIS. Their expression in the AIS and JXP-AIS is independent from transient axonal glycoprotein-1 (TAG-1)/Caspr2, in contrast to juxtaparanodes, and independent from PSD-93. Data from mice lacking the cytoskeletal linker protein 4.1B show that this protein is necessary to form the Caspr+ para-AIS barrier, ensuring the compartmentalization of Kv1 channels and the segregation of the AIS, para-AIS and JXP-AIS.

Conclusions: α Motor neurons have heterogeneous AISs, which underlie different spiking properties. However, they all have a para-AIS and a JXP-AIS contiguous to their AIS, where the myelin sheath begins, which might limit some AIS plasticity. Protein 4.1B plays a key role in ensuring the proper molecular compartmentalization of this hemi-node-type region.

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Voltage-gated potassium (Kv) channel distribution in the axon initial segments (AISs) in motor neurons (MNs). Triple immunostaining of Peripherin (A, F, K, P), ankyrin G (AnkG) (B, G, L, Q) and Kv channels: KCNQ2 (C), Kv1.1 (H, S), Kv1.2 (M) and Kvβ2 (R) (Kv and AnkG are merged in (D, I, N); Kv1.1 and Kvβ2 are merged in (T). (E, J, O) The mean immunofluorescence intensity profile (shown by the line) ± SEM from n = 6 AISs is shown for AnkG and Kv channels. For each AIS and each antibody, immunofluorescence intensities were normalized relative both to its maximum intensity along the AIS and to the length of the AIS. The beginning and the end of the AnkG+ AIS in (B, G, L, Q) are shown (also in E, J, O) by red arrowheads. Brackets indicate Kv channel expression domains. Scale bar = 5 μm.
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Figure 4: Voltage-gated potassium (Kv) channel distribution in the axon initial segments (AISs) in motor neurons (MNs). Triple immunostaining of Peripherin (A, F, K, P), ankyrin G (AnkG) (B, G, L, Q) and Kv channels: KCNQ2 (C), Kv1.1 (H, S), Kv1.2 (M) and Kvβ2 (R) (Kv and AnkG are merged in (D, I, N); Kv1.1 and Kvβ2 are merged in (T). (E, J, O) The mean immunofluorescence intensity profile (shown by the line) ± SEM from n = 6 AISs is shown for AnkG and Kv channels. For each AIS and each antibody, immunofluorescence intensities were normalized relative both to its maximum intensity along the AIS and to the length of the AIS. The beginning and the end of the AnkG+ AIS in (B, G, L, Q) are shown (also in E, J, O) by red arrowheads. Brackets indicate Kv channel expression domains. Scale bar = 5 μm.

Mentions: We next analyzed the MN AIS composition in terms of Kv channels. From this point, we focused our study on α MNs (identified either by their large soma or as NeuN+), because the following immunostainings were too weak in γ MNs to be properly analyzed. We found KCNQ2 expressed uniformly throughout the AnkG+ AIS in 100% of α MNs (Figure 4A-E; n = 37): its immunofluorescence intensity profile along the AIS closely matched that of AnkG (Figure 4E). We also found Kv1.1, Kv1.2 as well as Kvβ2 expressed at the AIS of 100% of α MNs (respectively n = 214, n = 48 and n = 34), but all three, in contrast to KCNQ2, were clearly absent from the proximal AIS (Figure 4F-T), as depicted by their immunofluorescence profile (Figure 4J, O). The three subunits appeared perfectly colocalized as shown for instance for Kv1.1 and Kvβ2 (Figure 4R-T), suggesting that heteromultimeric channels formed by the association of Kv1.1, Kv1.2 and Kvβ2 are present in the AIS, as previously shown in juxtaparanodes (JXP-nodes) [36]. Unlike Nav1.6, which was always found expressed at the proximal AIS, even when Nav1.1 was present (although then at a lower level relative to the distal AIS: Figure 3D, F), Kv1.1, Kv1.2 and Kvβ2 channels appeared completely absent from the proximal AIS, displaying a clear change in their expression level between the proximal and distal AIS, as shown by their fluorescence intensity profile (Figure 4H, J, M, O). MN AISs can thus be divided into different subcompartments along their proximodistal length, which express various combinations of Nav and Kv channels.


Characterization of the axon initial segment (AIS) of motor neurons and identification of a para-AIS and a juxtapara-AIS, organized by protein 4.1B.

Duflocq A, Chareyre F, Giovannini M, Couraud F, Davenne M - BMC Biol. (2011)

Voltage-gated potassium (Kv) channel distribution in the axon initial segments (AISs) in motor neurons (MNs). Triple immunostaining of Peripherin (A, F, K, P), ankyrin G (AnkG) (B, G, L, Q) and Kv channels: KCNQ2 (C), Kv1.1 (H, S), Kv1.2 (M) and Kvβ2 (R) (Kv and AnkG are merged in (D, I, N); Kv1.1 and Kvβ2 are merged in (T). (E, J, O) The mean immunofluorescence intensity profile (shown by the line) ± SEM from n = 6 AISs is shown for AnkG and Kv channels. For each AIS and each antibody, immunofluorescence intensities were normalized relative both to its maximum intensity along the AIS and to the length of the AIS. The beginning and the end of the AnkG+ AIS in (B, G, L, Q) are shown (also in E, J, O) by red arrowheads. Brackets indicate Kv channel expression domains. Scale bar = 5 μm.
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Related In: Results  -  Collection

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Figure 4: Voltage-gated potassium (Kv) channel distribution in the axon initial segments (AISs) in motor neurons (MNs). Triple immunostaining of Peripherin (A, F, K, P), ankyrin G (AnkG) (B, G, L, Q) and Kv channels: KCNQ2 (C), Kv1.1 (H, S), Kv1.2 (M) and Kvβ2 (R) (Kv and AnkG are merged in (D, I, N); Kv1.1 and Kvβ2 are merged in (T). (E, J, O) The mean immunofluorescence intensity profile (shown by the line) ± SEM from n = 6 AISs is shown for AnkG and Kv channels. For each AIS and each antibody, immunofluorescence intensities were normalized relative both to its maximum intensity along the AIS and to the length of the AIS. The beginning and the end of the AnkG+ AIS in (B, G, L, Q) are shown (also in E, J, O) by red arrowheads. Brackets indicate Kv channel expression domains. Scale bar = 5 μm.
Mentions: We next analyzed the MN AIS composition in terms of Kv channels. From this point, we focused our study on α MNs (identified either by their large soma or as NeuN+), because the following immunostainings were too weak in γ MNs to be properly analyzed. We found KCNQ2 expressed uniformly throughout the AnkG+ AIS in 100% of α MNs (Figure 4A-E; n = 37): its immunofluorescence intensity profile along the AIS closely matched that of AnkG (Figure 4E). We also found Kv1.1, Kv1.2 as well as Kvβ2 expressed at the AIS of 100% of α MNs (respectively n = 214, n = 48 and n = 34), but all three, in contrast to KCNQ2, were clearly absent from the proximal AIS (Figure 4F-T), as depicted by their immunofluorescence profile (Figure 4J, O). The three subunits appeared perfectly colocalized as shown for instance for Kv1.1 and Kvβ2 (Figure 4R-T), suggesting that heteromultimeric channels formed by the association of Kv1.1, Kv1.2 and Kvβ2 are present in the AIS, as previously shown in juxtaparanodes (JXP-nodes) [36]. Unlike Nav1.6, which was always found expressed at the proximal AIS, even when Nav1.1 was present (although then at a lower level relative to the distal AIS: Figure 3D, F), Kv1.1, Kv1.2 and Kvβ2 channels appeared completely absent from the proximal AIS, displaying a clear change in their expression level between the proximal and distal AIS, as shown by their fluorescence intensity profile (Figure 4H, J, M, O). MN AISs can thus be divided into different subcompartments along their proximodistal length, which express various combinations of Nav and Kv channels.

Bottom Line: We also identified in all α motor neurons a hemi-node-type organization, with a contactin-associated protein (Caspr)+ paranode-type, as well as a Caspr2+ and Kv1+ juxtaparanode-type compartment, referred to as a para-AIS and a juxtapara (JXP)-AIS, adjacent to the AIS, where the myelin sheath begins.We found that Kv1 channels appear in the AIS, para-AIS and JXP-AIS concomitantly with myelination and are progressively excluded from the para-AIS.Protein 4.1B plays a key role in ensuring the proper molecular compartmentalization of this hemi-node-type region.

View Article: PubMed Central - HTML - PubMed

Affiliation: INSERM UMRS 952, 9 Quai St Bernard, F-75005, Paris, France.

ABSTRACT

Background: The axon initial segment (AIS) plays a crucial role: it is the site where neurons initiate their electrical outputs. Its composition in terms of voltage-gated sodium (Nav) and voltage-gated potassium (Kv) channels, as well as its length and localization determine the neuron's spiking properties. Some neurons are able to modulate their AIS length or distance from the soma in order to adapt their excitability properties to their activity level. It is therefore crucial to characterize all these parameters and determine where the myelin sheath begins in order to assess a neuron's excitability properties and ability to display such plasticity mechanisms. If the myelin sheath starts immediately after the AIS, another question then arises as to how would the axon be organized at its first myelin attachment site; since AISs are different from nodes of Ranvier, would this particular axonal region resemble a hemi-node of Ranvier?

Results: We have characterized the AIS of mouse somatic motor neurons. In addition to constant determinants of excitability properties, we found heterogeneities, in terms of AIS localization and Nav composition. We also identified in all α motor neurons a hemi-node-type organization, with a contactin-associated protein (Caspr)+ paranode-type, as well as a Caspr2+ and Kv1+ juxtaparanode-type compartment, referred to as a para-AIS and a juxtapara (JXP)-AIS, adjacent to the AIS, where the myelin sheath begins. We found that Kv1 channels appear in the AIS, para-AIS and JXP-AIS concomitantly with myelination and are progressively excluded from the para-AIS. Their expression in the AIS and JXP-AIS is independent from transient axonal glycoprotein-1 (TAG-1)/Caspr2, in contrast to juxtaparanodes, and independent from PSD-93. Data from mice lacking the cytoskeletal linker protein 4.1B show that this protein is necessary to form the Caspr+ para-AIS barrier, ensuring the compartmentalization of Kv1 channels and the segregation of the AIS, para-AIS and JXP-AIS.

Conclusions: α Motor neurons have heterogeneous AISs, which underlie different spiking properties. However, they all have a para-AIS and a JXP-AIS contiguous to their AIS, where the myelin sheath begins, which might limit some AIS plasticity. Protein 4.1B plays a key role in ensuring the proper molecular compartmentalization of this hemi-node-type region.

Show MeSH
Related in: MedlinePlus