| Sumario: | Chandelier cells are a distinct subtype of GABAergic interneurons that form vertical arrays of presynaptic boutons targeting the axon initial segment of pyramidal neurons, thereby controlling the cell firing output. They are particularly abundant in the prefrontal cortex, where cholinergic inputs modulate cognitive functions and shape chandelier axonal development. However, the effects of the cholinergic system on chandelier activity in the adult brain remains largely unexplored. In this work, using a genetic intersectional strategy (VipR2-Pvalb-Ai65D) in adult mice, we selectively labelled chandelier cells in the secondary motor cortex. Immunohistochemistry and electrophysiology confirmed morphological and functional properties distinguishing them from basket cells. We found that these chandelier cells exhibit an inward current in response to nicotinic acetylcholine receptor agonists. Pharmacological blockade and fluorescent in situ hybridization revealed that this effect is primarily mediated by heteromeric nicotinic acetylcholine receptors containing ?2 subunits, with a smaller contribution from ?3 subunits. Optogenetic stimulation of basal forebrain cholinergic axons evoked inward currents in chandelier cells, confirming a functional cholinergic drive. To elucidate the role of chandelier cells in prefrontal cortical circuitry, we performed in vivo two-photon calcium imaging in awake mice using genetically encoded calcium indicators. We found that chandelier cells exhibit collective, synchronized activity during arousal. Importantly, the application of nAChR antagonists significantly reduced locomotion-associated ChC activity. Finally, chemogenetic manipulation revealed that altering chandelier activity does not globally shift pyramidal neuron activity, but instead induces heterogeneous and state-dependent changes across excitatory ensembles. Together, our findings establish prefrontal chandelier cells as a distinct population of fast-spiking interneurons, strongly modulated by cholinergic inputs via heteromeric nAChRs, and engaged during arousal to fine-tune, rather than broadly suppress, cortical pyramidal networks.
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