Acetylcholine (ACh) exerts diverse and powerful effects on animal behavior and underlying cortical neural dynamics. However, identifying the cellular and circuit substrates mediating these processes has proved challenging due to the many targets of ACh action and the lack of specific tools. ACh is thought to act on local cortical circuit components, specifically interneurons, to indirectly influence pyramidal neuron dynamics. We hypothesize that direct cholinergic neuromodulation of pyramidal neurons dendrites is an important new locus for the effects of ACh on cortical dynamics and behavior. By leveraging a new genetically targeted pharmacological tool with unprecedented specificity, we will causally test the contribution of AChR-dependent dendritic mechanisms to a cortical sensorimotor computation.!Our preliminary evidence shows that muscarinic acetylcholine receptors (mAChRs) potently modulate the excitability of distal apical trunk dendrites in layer 5 cortical pyramidal neurons (L5 PNs). These dendrites exhibit an active supralinear mechanism that can drive high frequency somatic spiking during coincident “bottom-up” and “top-down” cortical input. L5 PN trunk dendrites are therefore well positioned to implement a canonical cortical computation for combining multiple inputs. In the mouse barrel cortex, bottom-up sensory information is combined with top-down motor input via this subcellular coincidence detection mechanism to produce a whisker object localization signal. We will use this system to test a novel role for ACh in cortical function by characterizing the effect of mAChR activation on L5 PN dendritic integration (Aim 1) and identifying its ion channel mechanism (Aim 2). We will then employ a novel genetically-targeted pharmacology strategy with unprecedented specificity to causally test the contributions of mAChR-dependent dendritic mechanisms to a cortical sensorimotor computation during behavior (Aim 3). These experiments will establish a new pathway linking a single neuromodulator – and its ion channel target(s) in a genetically defined L5 PN cell type – to cellular processing, circuit computation, and behavior, providing critical insight into how ACh modulates brain function. !