PROJECT SUMMARY Down syndrome (DS) is the most commonly diagnosed chromosomal condition and the most common genetic cause of intellectual disability in the US. DS affects a range of behavioral domains in children, including motor and cognitive function. While atypical cognitive processing has been well studied in DS, locomotor dysfunction is relatively understudied. Clinical assessments indicate a range of locomotor deficits in DS, as well as slower adaptive control. Longitudinal data also indicates altered gait evolution from childhood to adulthood. Cerebellar pathology has been consistently observed in DS, and is thought to contribute to dysfunction in locomotor and adaptive motor skills. Studies in animal models of DS have also indicated deficient cerebellar processing. However, the specific pathways underlying locomotor deficits and the cerebellar circuits that are disrupted in DS remain poorly understood. Defining specific abnormalities in motor behavior, and identifying the brain regions and neurons which are functionally involved will provide the basis for developing potential therapies for treating motor problems in individuals with DS. The main goal of this proposal is to identify specific alterations in the circuitry of the cerebellum that result in locomotor dysfunction in DS. Our preliminary data show locomotor miscoordination, adaptive motor learning deficits, and cerebellar synaptic alterations in the Ts65Dn mouse model of DS. To quantify locomotor behavior, we used the ErasmusLadder, an advanced tool capable of measuring locomotor coordination and adaptive cerebellar learning. Our analysis in postnatal Ts65Dn mice shows that Purkinje cells (PCs), which are the sole output of the cerebellar cortex, receive fewer excitatory synapses from climbing fibers (CFs) than normal. This finding is significant, as cerebellar-dependent learning depends on strong monosynaptic excitatory input from CFs onto PC dendrites. Based on our data, we hypothesize that locomotor dysfunction and adaptive motor deficits in the Ts65Dn mouse model of DS are caused by disruption of CF input to PCs. To test this hypothesis, in Aim 1 we will define changes in PC circuitry that are linked to abnormal synaptic input to PCs and to locomotor learning deficits in Ts65Dn mice. We will analyze locomotor dysfunction and identify molecular changes in cerebellar circuitry to define potential synaptic alterations in the cerebellar cortex. In Aim 2, we will establish the precise correlation between pathophysiological changes in PC activity and locomotor abnormalities in freely-behaving animals, and determine whether enhancing excitatory input to PCs will restore locomotor function in Ts65Dn mice. We will use an advanced technique established in our lab that employs GCaMP6f fiber photometry in order to time- lock PC activity in the cerebellum to ErasmusLadder behavioral data. We will attempt at rescuing abnormalities in PC activity and locomotor behavior in Ts65Dn mice by spec...