Abundant extrasynaptic expression of α3β4-containing nicotinic acetylcholine receptors in the medial habenula-interpeduncular nucleus pathway in mice

Nicotinic acetylcholine receptors (nAChRs) in the medial habenula (MHb)-interpeduncular nucleus (IPN) pathway play critical roles in nicotine-related behaviors. This pathway is particularly enriched in nAChR alpha 3 and beta 4 subunits, both of which are genetically linked to nicotine dependence. However, the cellular and subcellular expression of endogenous alpha 3 beta 4-containing nAChRs remains largely unknown because specific antibodies and appropriate detection methods were unavailable. Here, we successfully uncovered the expression of endogenous nAChRs containing alpha 3 and beta 4 subunits in the MHb-IPN pathway using novel specific antibodies and a fixative glyoxal that enables simultaneous detection of synaptic and extrasynaptic molecules. Immunofluorescence and immunoelectron microscopy revealed that both subunits were predominantly localized to the extrasynaptic cell surface of somatodendritic and axonal compartments of MHb neurons but not at their synaptic junctions. Immunolabeling for alpha 3 and beta 4 subunits disappeared in alpha 5 beta 4-knockout brains, which we used as negative controls. The enriched and diffuse extrasynaptic expression along the MHb-IPN pathway suggests that alpha 3 beta 4-containing nAChRs may enhance the excitability of MHb neurons and neurotransmitter release from their presynaptic terminals in the IPN. The revealed distribution pattern provides a molecular and anatomical basis for understanding the functional role of alpha 3 beta 4-containing nAChRs in the crucial pathway of nicotine dependence.