Dual cholinergic mechanisms for sculpting striatal dopamine in vivo

Striatal dopamine (DA) and acetylcholine constitute a computationally powerful neuromodulatory dyad that orchestrates action selection, motivational vigor, and reward learning. Striatal cholinergic interneurons (CINs) synapse onto DA axons and stimulate DA release via β₂-containing nicotinic receptors (β₂-nAChRs), providing a local modulatory channel distinct from midbrain-derived spikes. Yet whether this mechanism operates in vivo and how it contributes to striatal computations, orthogonal to those facilitated by prediction errors encoded in the DA somata, remains unresolved. Using spatiotemporally precise optogenetic stimulation of CINs paired with ultrafast DA imaging in the dorsal striatum, we identify a biphasic mode of CIN-evoked DA release in awake mice. CIN activation evoked an ultra-fast β₂-nAChR-dependent DA transient peaking within ~40 ms that requires millimeter-scale CIN synchrony and is systematically obscured by conventional imaging bandwidths or slow DA sensor kinetics. A second, delayed DA elevation peaking at ~180 ms persisted despite β₂-nAChR blockade and is mediated by muscarinic receptors, α₇- and α₆*-nAChRs, and a non-cholinergic CIN messenger, revealing a previously unrecognized polysynaptic CIN mechanism. Our observations reconcile discrepancies between slice and in vivo studies and show that CINs engage DA release through two mechanistically distinct pathways: (i) an ultra-fast, monosynaptic mode provides a spatially expansive DA signal ideally positioned to reconfigure striatal microcircuits globally, and (ii) a slower, spatially flexible polysynaptic DA mode suited for sustaining ongoing decisions. This dual architecture expands the computational repertoire of striatal DA dynamics in behavioral and cognitive control.