PROJECT SUMMARY/ABSTRACT Rett syndrome (RTT) is the leading cause of severe intellectual disability in girls and women. Individuals with RTT develop typically until 6-18 months, when autism-like behaviors and deficits in purposeful hand use and speech start to develop. Loss-of-function mutations in the transcriptional regulator methyl-CpG-binding protein 2 (MECP2) occur in >95% of RTT cases. Initially, researchers regarded MeCP2 deficiency in neurons as the exclusive cause of RTT. However, recent studies have revealed that glia have a pathological role in RTT etiology. Studies have not yet uncovered the underlying cellular mechanisms of glial cell dysfunction in Mecp2 rodent models or the impact of this dysfunction on brain function. In this study, we propose to examine the contribution of a specific glial subtype, Bergmann glia cells (BGs) in the cerebellum, to the motor and social deficits in RTT. We will focus on BGs because (1) cerebellar disruption likely contributes to the motor and social deficits in RTT and (2) BGs are the major glial subtype in the cerebellum. We propose to characterize cerebellar function in conditional knockout (cKO) mice that lack Mecp2 in BGs using a repertoire of approaches, including intracellular whole-cell recordings, in vivo recordings of single units and local field potentials, in vivo and ex vivo imaging of genetically-encoded Ca2+, glutamate, and dopamine (DA) sensors (GCaMP7, iGluSnFr, and dLight1.1, respectively), optogenetics, chemogenetics, near super-resolution confocal microscopy, and behavioral assessments using unbiased machine-learning approaches. Our preliminary results indicate deficits in BGs from Mecp2-deficient mice, including higher expression levels of the GluA1 subunit of AMPA receptors (AMPARs), and depolarized membrane potentials. In addition, we have preliminary evidence of DAergic modulation of BGs through the activation of D1 receptors (D1Rs) activated by either electrical or optogenetic stimulation of DAergic inputs to the cerebellum. Of high relevance to motor and social symptoms in RTT, the expression levels of D1Rs, which modulate GluA1 surface insertion, are higher in Mecp2 KO mice. We hypothesize that BGs in the cerebellum have a pathophysiological role in motor and social deficits in Mecp2-deficient mice. We propose two Aims: (1) Define cerebellar BG dysfunction in Mecp2 cKO mice;; (2) Characterize DAergic modulation of cerebellar BGs in Mecp2 cKO mice. The proposed studies will elucidate the consequences of impaired DAergic modulation of astrocyte function on cerebellar-dependent behaviors, which will have broad implications not only for RTT but also for other brain disorders ass...