PROJECT SUMMARY As animals interact with their environment, they build and accrue sound associations with behaviorally relevant outcomes. The mammalian auditory system can be modified by experience and by behavioral context and supports the ability to build these associations. The auditory cortex (AC) supports the ability for animals to learn that particular sounds can signal rewards. The neural responses in the primary auditory cortex (A1) can be re-shaped by audiomotor learning, taking the form of changes at the single-neuron and population level. This learning-induced plasticity decreases after animals are trained at expert levels of behavioral performance for several weeks until the cortical map appears to renormalize and neural responses revert to a near pre- learning state. The plasticity that emerges across learning is not preserved in the cortex during the overtraining phase, yet animals are able to retain task performance. The overall objective of this proposal is to if A1 tutors or offloads these combined representations of sound and action to another pathway for long-term use, which supports long term execution of the task and the renormalization of A1. Our central hypothesis is that the A1 tutors subcortical auditory regions—the medial geniculate body (MGB) and the inferior colliculus (IC)— which subsequently store audiomotor associations. I will address this question in two aims. In Aim 1, I will determine the spatiotemporal dynamics of plasticity in the IC, MGB, and AC across learning and overtraining. In Aim 2, I will identify the causal contributions of the MGB and the IC at the expert level. To do this, I will train mice on an auditory go/no-go task where mice learn to lick to a pure tone for a water reward (S+) and withhold licking to another tone (S-) to avoid a timeout. We use large-scale, two-photon mesoscopic imaging to monitor neural activity in IC neurons, MGB axons, and A1 neurons over the entire course of learning and overtraining (28 days). I developed a new surgical preparation which allows the implantation of a single cranial window over the IC and the AC to enable simultaneous calcium imaging of both regions and the feedforward projections from the MGB to the AC. By tracking the same cell bodies and axons across a month of training, we examine the sequence of stimulus-related and non-stimulus related plasticity and the nature of how learning and overtraining impact these processes across the sensory hierarchy. Using a combination of state-of-the-art optical tools, temporally precise optogenetics, large-scale calcium imaging, and custom computational analyses and tools, these experiments will help us understand the nature of cortical-subcortical interactions that support auditory learning and memory consolidation along three processing stations in the central auditory system.