A number of studies over the past decade have shown that astrocytes, the supportive cells of the brain, play important roles in synaptic transmission including regulating the strength of both excitatory and inhibitory synapses. A major challenge for the future is to define the role of astrocytes in complex tasks, such as functional hyperemia and sensory processing, as well as their contribution to acute and degenerative diseases of the nervous system. Multiphoton imaging approaches are ideally suited to study electrically non-excitable astrocytes. We here discuss novel in vivo studies aimed at defining the role of astrocytes in normal and pathological brain function. With a better understanding of the role astrocytes play in information processing and regulation of the brain microenvironment in vivo, and the understanding that astrocytes are heavily implicated in the pathology of many diseases such as epilepsy, Alzheimer's and Parkinson's diseases, astrocytes provide a promising target for future drug therapy approaches.