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The medial habenula (MHb) receives afferents from the triangular septum and the medial septal complex, projects efferents to the interpeduncular nucleus (IPN) in the midbrain to regulate dopamine and serotonin levels, and is implicated in stress, depression, memory, and nicotine withdrawal syndrome. We previously showed that the cell adhesion molecule nectin‐2α is localized at the boundary between adjacent somata of clustered cholinergic neurons and regulates the voltage‐gated A‐type K+ channel Kv4.2 localization at membrane specializations in the MHb. This adhesion apparatus, named nectin‐2α spots, is not associated with the nectin‐binding protein afadin or any classic cadherins and their binding proteins p120‐catenin and β‐catenin. We showed here that nectin‐2α was additionally localized at cholinergic neuron dendrites in synaptic regions of the MHb. The genetic ablation of nectin‐2 reduced the number of synapses in the MHb without affecting their morphology. Nectin‐2α was associated with afadin, cadherin‐8, p120‐catenin, β‐catenin, and αN‐catenin, forming puncta adherentia junctions (PAJs). Nectin‐2α was observed in the IPN, but not in the triangular septum or the medial septal complex. The genetic ablation of nectin‐2 did not affect synapse formation in the IPN. These results indicate that nectin‐2α forms two types of adhesion apparatus in the MHb, namely nectin‐2α spots at neighboring somata and PAJs at neighboring dendrites, and that dendritic PAJs regulate synapse formation in the MHb. [ABSTRACT FROM AUTHOR]

In the cerebellum, GluD2 is exclusively expressed in Purkinje cells, where it regulates synapse formation and regeneration, synaptic plasticity, and motor learning. Delayed cognitive development in humans with GluD2 gene mutations suggests extracerebellar functions of GluD2. However, extracerebellar expression of GluD2 and its relationship with that of GluD1 are poorly understood. GluD2 mRNA and protein were widely detected, with relatively high levels observed in the olfactory glomerular layer, medial prefrontal cortex, cingulate cortex, retrosplenial granular cortex, olfactory tubercle, subiculum, striatum, lateral septum, anterodorsal thalamic nucleus, and arcuate hypothalamic nucleus. These regions were also enriched for GluD1, and many individual neurons coexpressed the two GluDs. In the retrosplenial granular cortex, GluD1 and GluD2 were selectively expressed at PSD‐95‐expressing glutamatergic synapses, and their coexpression on the same synapses was shown by SDS‐digested freeze‐fracture replica labeling. Biochemically, GluD1 and GluD2 formed coimmunoprecipitable complex formation in HEK293T cells and in the cerebral cortex and hippocampus. We further estimated the relative protein amount by quantitative immunoblotting using GluA2/GluD2 and GluA2/GluD1 chimeric proteins as standards for titration of GluD1 and GluD2 antibodies. Intriguingly, the relative amount of GluD2 was almost comparable to that of GluD1 in the postsynaptic density fraction prepared from the cerebral cortex and hippocampus. In contrast, GluD2 was overwhelmingly predominant in the cerebellum. Thus, we have determined the relative extracerebellar expression of GluD1 and GluD2 at regional, neuronal, and synaptic levels. These data provide a molecular–anatomical basis for possible competitive and cooperative interactions of GluD family members at synapses in various brain regions. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]