ABSTRACT Rett Syndrome (RTT) is a rare X-linked developmental brain disorder due to de novo pathogenic variants in MECP2 and mainly affecting girls. Notably, RTT individuals reach typical developmental milestones in the first 6-18 months of life, followed by stagnation and then regression of acquired skills. The severe cognitive delays, deficits in sensory processing and dysregulated behavioral states profoundly impact both patient and family quality of life. We still do not know when and how autonomic and central brain networks begin to derail from the neurotypical developmental trajectory, nor we have effective treatments targeting these impairments. Hence, there is an urgent need for objective, quantitative, non-invasive, and translational biomarkers for early assessment of cognition and behavioral states in RTT, their progression over time and response to therapeutic interventions. Our goals are to establish 1) spontaneous pupil and heart rate (HR) fluctuations as new biomarkers for RTT, 2) how arousal impacts the progression of RTT cortical pathophysiology and 3) develop targeted interventions. We will address these challenges using a multi-level circuit approach both in RTT girls and awake Mecp2 female heterozygote mice during the progression of the disorder. The proposed work will refine and establish spontaneous pupil and HR fluctuations as highly translational biomarkers to track autonomic nervous system function, while dissecting how and when neuromodulation impacts sensory processes in RTT. Together these approaches will allow the development of new circuit-based therapies in patients. Our results will be pave the way to future studies of RTT related disorders such as MECP2 duplication, CDKL5 deficiency disorder and FOXG1 syndrome.