Molecular brake on firing pattern transitions in MHb^ChAT neurons to mediate nicotine-withdrawal-induced anxiety

Cholinergic neurons exhibit distinct firing patterns underlying diverse physiological and pathological states, but the mechanisms governing their dynamic switching, particularly in negative emotional contexts, remain unclear. Here, we demonstrate that medial habenula cholinergic (MHb^ChAT) neurons transition from tonic to burst firing during nicotine withdrawal, driving anxiety-like behaviors in mice. Integrating transcriptomics, electrophysiology, and genetic manipulation, we identified the RNA-binding protein pumilio 1 (Pum1) as a critical brake on this switch. Pum1 binds Cacna1g mRNA (encoding Cav3.1) at nucleotides 6,498-6,501, promoting its decay. MHb^ChAT neurons comprise two subpopulations: burst-firing Pum1^- and tonic-firing Pum1⁺ neurons. Withdrawal downregulates Pum1, derepressing Cav3.1 to induce pathological bursting. Genetic or pharmacological suppression of Cav3.1, or Pum1 overexpression, rescues burst firing and anxiety-like behaviors. Our study unveils MHb^ChAT neurons' burst firing as a causal driver of anxiety and reveals the Pum1-Cav3.1 axis as a master regulator of firing plasticity, offering a potential targeted therapeutic strategy for cholinergic dysfunction-related disorders.