Our long-term goal is to determine how cholinergic signaling drives adaptive sound processing in the auditory midbrain and how this computational flexibility is altered in auditory gating disorders, hearing loss, and aging. As the midbrain link between the auditory brainstem and forebrain, the inferior colliculus (IC) is a critical processing center that influences which auditory information gets presented to the forebrain. Computations in the IC are modulated by cholinergic projections from brainstem cholinergic systems, including a dense projection from the pedunculopontine tegmentum (PPT). PPT neurons convey information about states of arousal, rewards, and the salience of sensory stimuli, including sounds. While previous in vivo studies have shown that cholinergic drugs elicit diverse effects on IC neurons and that cholinergic signaling can drive plasticity in the IC, the cellular and synaptic logic of cholinergic modulation in the IC have remained largely unknown. This gap in knowledge has persisted due to the technical obstacles created by the diversity of neuron types and convergence of ascending and descending pathways in the IC. This is a critical gap in knowledge because understanding how cholinergic signaling influences specific populations of IC neurons and their synaptic inputs is a prerequisite to understanding how cholinergic signaling links changes in internal and external states to changes in auditory processing. We have recently overcome previous obstacles to progress by developing molecular genetic techniques to label and manipulate specific populations of IC neurons and their sources of synaptic input. Based on our past and preliminary data, the current proposal tests the overall hypothesis that the effects of cholinergic signaling in the IC are largely determined by two factors: (1) the muscarinic and nicotinic acetylcholine receptors expressed in specific populations of IC neurons and their synaptic inputs and (2) the subcellular location and timing of acetylcholine release. We propose four aims to test this hypothesis. In Aim 1, we use pharmacology and patch clamp recordings targeted to fluorescently labeled populations of IC neurons to determine cellular mechanisms of cholinergic signaling in distinct populations of excitatory and inhibitory IC neurons. In Aim 2, we combine optogenetic circuit mapping with pharmacology and patch clamp recordings to determine how muscarinic signaling modulates specific sources of synaptic input to the IC. In Aim 3, we use electron microscopy to identify ultrastructural relationships between cholinergic boutons and IC neurons. In Aim 4, we use two-photon Ca2+ imaging in awake mice to determine how and when cholinergic axons from the PPT respond to auditory stimuli. The proposed studies will provide a functional and ultrastructural map of how cholinergic signaling alters excitability in distinct populations of IC neurons and their synaptic inputs. The fundamental insights gained will be essen...