Blood oxygen level-dependent (BOLD) functional brain imaging is a valuable non-invasive tool widely used in studies of brain development in health and disease, which relies on blood flow and oxygenation state to provide insights into brain function. Intriguingly, functional imaging studies in the newborn developing brain often report unexplained variation and early inverted hemodynamic responses. There is a gap in knowledge to explain the discrepancy in the newborn hemodynamic signals that needs to be addressed for a better understanding of functional imaging data. The main goal of this project is to identify the origins of this shift. This will be achieved through the combination of a multidisciplinary team with cutting-edge methodologies to study the neonatal brain development in the living mouse. 2BOLD will implement specific aims that leverage innovative non-invasive imaging with a mechanistic insight approach. In Aim 1, we will longitudinally track, using suited pre-clinical imaging, the co-development of hemodynamic, vascular, and metabolic activity. Here, we aim to resolve the spatiotemporal developmental switch from negative to positive responses. In Aim 2, we will use in vivo 2-photon microscopy, novel transgenic animals and optogenetic manipulation to elucidate if the emergence of perivascular astrocytic endfeet is linked with cerebrovascular tone development and neurovascular coupling. Lastly, in Aim 3, live imaging will be complemented by comprehensive proteomic and metabolomic profiling of cerebrovascular maturation associated with the hemodynamic changes. This groundbreaking project will capture the dynamics of cerebrovascular events and hemodynamic signatures with unprecedented detail in the intact mammalian neonatal brain. This advance may pave the way for mechanistic studies on how injury or other adverse processes might derail normal development, with an impact on the diagnosis and treatment of neonatal brain pathologies, such as cerebral palsy.