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

Abstract Chandelier cells (ChCs) are a highly specialized subtype of GABAergic interneurons and one of the most distinctive elements of cortical circuitry, exerting powerful and strategic control over pyramidal neuron output by selectively innervating their axon initial segment. They are particularly abundant in the prefrontal cortex, where cholinergic inputs modulate cognitive functions and shape ChC axonal development, but the way in which the cholinergic system—a master modulator of attention and arousal—regulates these cells in the adult brain has long remained unexplored. In this study, by employing an intersectional genetic strategy in adult mice, we reveal that ChCs in the secondary motor cortex are direct targets of cholinergic modulation. Through patch-clamp recordings and functional imaging, we demonstrate that acetylcholine persistently activates ChCs via heteromeric nicotinic receptors containing the β2 subunit, triggering robust depolarization and a significant increase in intrinsic excitability. This regulation does not rely on fast synaptic transmission; instead, it arises from a diffuse and sustained cholinergic signaling mode, orchestrated from the basal forebrain. Intriguingly, our in vivo observations show that ChC activity is positively correlated with behavioral markers of high arousal, such as locomotion and pupil dilation—a signature of activity that diminished upon the blockade of nicotinic receptors. Our findings strongly suggest that ChCs serve as a link between global arousal and local cortical control, thereby offering deeper insights into the mechanisms of state-dependent information processing. Competing Interest Statement The authors have declared no competing interest. Footnotes We have uploaded a revised version of our manuscript to incorporate new experiments and a refined conceptual framework. In this version, we introduce physostigmine experiments that demonstrate how acetylcholinesterase inhibition produces a gradual, sustained increase in chandelier cell excitability, fully dependent on nicotinic receptors, providing evidence for nicotinic volume transmission as a tonic regulator of these interneurons. We also clarify the contribution of specific nicotinic receptor subunits, strengthen the link between chandelier cell recruitment and behavioral markers of arousal, and streamline the narrative to emphasize their role as a bridge between global cholinergic arousal signals and local cortical output control. Finally, we have polished the text, updated the figures and legends, and expanded the discussion to better integrate our findings with current models of state‑dependent cortical computation.