Cholinergic Modulation of Chandelier Cells via Heteromeric Nicotinic Receptors in Prefrontal Cortex

Abstract Chandelier cells (ChCs) are a distinct subtype of GABAergic interneurons that form vertical arrays of presynaptic boutons targeting the axon initial segment (AIS) of pyramidal neurons (PNs). They are particularly abundant in prefrontal cortex, where cholinergic inputs modulate cognitive functions and shape ChC axonal development. However, the effects of the cholinergic system on ChC activity in the adult brain remain largely unexplored. Here, using a genetic intersectional strategy ( VipR2-Pvalb-Ai65 ) in adult mice, we selectively labeled ChCs in the secondary motor cortex (M2). Immunohistochemistry and electrophysiology confirmed morphological and functional properties distinguishing them from basket cells. We found that these ChCs exhibit an inward current in response to nicotinic acetylcholine receptor (nAChR) agonists. Pharmacological blockade and fluorescent in situ hybridization revealed that this effect is primarily mediated by heteromeric nAChRs containing β2 subunits, with a smaller contribution from α3 subunits. Optogenetic stimulation of basal forebrain (BF) cholinergic axons evoked inward currents in ChCs, confirming a functional cholinergic drive. Furthermore, in vivo two-photon calcium imaging revealed a strong correlation between ChC activity and behavioral markers of arousal, including locomotion and pupil dilation. Importantly, the application of nicotinic receptor antagonists significantly diminished ChC activity during movement. Together, these results indicate that ChCs are modulated by cholinergic input via heteromeric nAChRs, emphasizing their importance in facilitating state-dependent cortical dynamics.