Multiplexed calcium imaging of single-synapse activity and astroglial responses in the intact brain.

All-optical registration of neuronal and astrocytic activities within the intact mammalian brain has improved significantly with recent advances in optical sensors and biophotonics. However, relating single-synapse release events and local astroglial responses to sensory stimuli in an intact animal has not hitherto been feasible. Here, we present a multiplexed multiphoton excitation imaging approach for assessing the relationship between presynaptic Ca entry at thalamocortical axonal boutons and perisynaptic astrocytic Ca elevations, induced by whisker stimulation in the barrel cortex of C57BL/6 mice. We find that, unexpectedly, Ca elevations in the perisynaptic astrocytic regions consistently precede local presynaptic Ca signals during spontaneous brain activity associated with anaesthesia. The methods described here can be adapted to a variety of optical sensors and are compatible with experimental designs that might necessitate repeated sampling of single synapses over a longitudinal behavioural paradigm.