Sensorimotor integration in the cerebellum is essential for refining motor output. Much of this integration occurs at the initial stage of cerebellar processing, in the granule cell layer where mossy fibers carrying diverse sensory and motor information converge. While these computations have been thought to occur through rigid, anatomically defined circuits, recent evidence suggests that granule cell layer integration can be contextually modified. Neuromodulators represent a strong candidate for such regulation, and anatomical studies have revealed prominent cholinergic and serotonergic projections into the cerebellar granule cell layer. However, it is unknown how these neuromodulators act at the cellular and circuit level to control sensory and motor integration. Our preliminary data reveal that Golgi cells, interneurons that provide the sole source of inhibition to the granule cell layer, express receptors for both acetylcholine (ACh) and serotonin (5-HT). We find that these neuromodulators bi-directionally regulate the excitability of Golgi cells: ACh suppresses Golgi cell spiking while 5-HT elevates spiking. In addition, we find that granule cells are depolarized by ACh. This suggests that ACh may generally act to increase excitability in the granule cell layer. Using a combination of modern physiological, genetic and anatomical approaches in the mouse, we will test the following aims: In Aim 1 we will use an in vitro brain slice preparation to identify the sites of ACh and 5-HT receptor expression on the major cell classes of the granule cell layer: the granule cells, Golgi cells and mossy fibers. Using targeted application of neuromodulatory agonists and specific pharmacology, we will determine how these neuromodulators directly impact cellular excitability both acutely and after prolonged exposure. In Aim 2, we will use retrograde tracing to identify the sources of cerebellar cholinergic and serotonergic inputs. This will allow us to identify whether these neuromodulatory inputs are part of a larger, brain-wide system, and under what conditions they are active. Localizing the afferent neuromodulatory nuclei will also allow viral delivery of optogenetic proteins, and thus investigation of the effect of endogenously released neuromodulators on the intact granule cell circuit. In particular, we will test the hypothesis that ACh acts to increase excitability in the granule cell layer while 5-HT acts to decrease it. Then in Aim 3, we will test how these neuromodulatory effects on excitability regulate granule cell layer integration of sensory and motor input in vivo. We will use multi-unit electrophysiology and two-photon imaging to determine how activation of cholinergic and serotonergic inputs alter the activity of the population of granule cells. In particular, we will present sensory stimuli with graded intensity to test the hypothesis that ACh and 5-HT change the gain of the granule cell network. Together, these experiments will reveal th...