Localization of nectin‐2α at the boundary between the adjacent somata of the clustered cholinergic neurons and its regulatory role in the subcellular localization of the voltage‐gated A‐type K⁺ channel Kv4.2 in the medial habenula

Abstract

The medial habenula (MHb), implicated in stress, depression, memory, and nicotine withdrawal syndromes, receives septal inputs and sends efferents to the interpeduncular nucleus. We previously showed that the immunoglobulin-like cell adhesion molecules (CAMs) nectin-2α and nectin-2δ are expressed in astrocytes in the brain, but their expression in neurons remains unknown. We showed here by immunofluorescence microscopy that nectin-2α, but not nectin-2δ, was prominently expressed in the cholinergic neurons in the developing and adult MHbs and localized at the boundary between the adjacent somata of the clustered cholinergic neurons where the voltage-gated A-type K channel Kv4.2 was localized. Analysis by immunoelectron microscopy on this boundary revealed that Kv4.2 was localized at the membrane specializations (MSs) with plasma membrane darkening in an asymmetrical manner, whereas nectin-2α was localized on the apposed plasma membranes mostly at the outside of these MSs, but occasionally localized at their edges and insides. Nectin-2α at this boundary was not colocalized with the nectin-2α-binding protein afadin, other CAMs, or their interacting peripheral membrane proteins, suggesting that nectin-2α forms a cell adhesion apparatus different from the Kv4.2-associated MSs. Genetic ablation of nectin-2 delayed the localization of Kv4.2 at the boundary between the adjacent somata of the clustered cholinergic neurons in the developing MHb. These results revealed the unique localization of nectin-2α and its regulatory role in the localization of Kv4.2 at the MSs in the MHb.

Nectin‐2α is localized at cholinergic neuron dendrites and regulates synapse formation in the medial habenula

“…The results shown are representative of three independent experiments et al, 2015). We previously showed that nectin-2α localized at the boundary between adjacent somata of clustered cholinergic neurons constitutes nectin-2α spots with which none of l-afadin, s-afadin, cadherins, and their binding proteins is associated (Shiotani et al, 2018). In contrast, the nectin-2α-mediated adhesion apparatus identified here at the contact sites between F I G U R E 1 6 Nectin-2α colocalization with l-afadin, cadherin-8, αN-catenin, and F-actin in the S1 region of the MHb.…”

“…We previously showed by immunofluorescence microscopy on the adult MHb using the anti-nectin-2α/δ mAb that two different staining patterns of the immunofluorescence signal for nectin-2α are observed: one is a linear pattern and the other is a punctate pattern (Shiotani et al, 2018). Consistent with these previous observations, the signal for nectin-2α with the linear pattern was observed mainly in the MHbI and the MHbCv of the adult MHb (Figure 2a,b, arrowheads), whereas that with the punctate pattern was observed in the whole regions of the MHb (Figure 2a,b, arrows).…”

“…We thereafter showed that nectin‐2α, but not nectin‐2δ, is prominently expressed in the adult MHb (Shiotani et al, 2018). Nectin‐2α is expressed in cholinergic neurons in the developing and adult MHbs and localized both at the boundary between adjacent somata of clustered cholinergic neurons and in synaptic regions.…”

“…The physiological function of MSs in the MHb remains unknown. The analysis by immunoelectron microscopy on this boundary revealed that Kv4.2 is localized at MSs with plasma membrane darkening in an asymmetrical manner, whereas nectin‐2α is localized on apposed plasma membranes mostly at the outside of these MSs, but occasionally localized at their edges and insides (Shiotani et al, 2018). Nectin‐2α at this boundary is not colocalized with the nectin‐binding protein afadin, any classic cadherins, or their binding proteins p120‐catenin and β‐catenin (Shiotani et al, 2018).…”

“…The analysis by immunoelectron microscopy on this boundary revealed that Kv4.2 is localized at MSs with plasma membrane darkening in an asymmetrical manner, whereas nectin‐2α is localized on apposed plasma membranes mostly at the outside of these MSs, but occasionally localized at their edges and insides (Shiotani et al, 2018). Nectin‐2α at this boundary is not colocalized with the nectin‐binding protein afadin, any classic cadherins, or their binding proteins p120‐catenin and β‐catenin (Shiotani et al, 2018). The nectin‐2α‐mediated adhesion apparatus was thus different from Kv4.2‐associated MSs but was similar to nectin‐1 spots originally identified at the contacts between mitral cell lateral dendrites in the olfactory bulb (Inoue et al, 2015) and named nectin‐2α spots (Shiotani et al, 2018).…”