Vision is a critical sensory modality that we depend on to navigate through the world and understand our surroundings. Disorders of the visual system lead to severe interpersonal deficits and to economic immiseration. Although much progress has been made describing the cellular basis of visual perception, little is known about the connectivity of brain circuits that process visual information, and even less is known about how this connectivity changes over time. I propose to leverage cutting-edge robotic and optical technologies to clarify how neuronal ensembles (coactive groups of cells) are connected in the primary visual cortex (V1), the neocortical region where visual perception arises. Ensembles in V1 exhibit activity patterns with reproducible spatial and temporal structures which define the functional vocabulary of cortical microcircuits. It has been recently shown that the activation of ensembles is necessary and sufficient for visual perception. The Hebbian hypothesis suggests that neurons that are repeatedly coactive over time (ensembles) are likely to be more strongly synaptically connected to one another than to neurons outside of the ensemble. I will develop a high-throughput tool to test this Hebbian hypothesis of preferential synaptic connectivity within ensembles using robotic electrophysiology and holographic optogenetic stimulation (Aim 1). I will then describe the functional and structural changes of cortical microcircuits during visual learning using chronic two-photon calcium imaging of neurons in the mouse visual cortex, in relation to the Hebbian hypothesis of synaptic plasticity and learning (Aim 2). Successful completion the current project will establish a structural link between ensemble activity and the activity of the brain during visual learning, yielding inroads towards a more complete understanding of visual processing, a prerequisite to addressing the dearth of effective treatment options for blindness.