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 Ca²⁺ entry at thalamocortical axonal boutons and perisynaptic astrocytic Ca²⁺ elevations, induced by whisker stimulation in the barrel cortex of C57BL/6 mice. We find that, unexpectedly, Ca²⁺ elevations in the perisynaptic astrocytic regions consistently precede local presynaptic Ca²⁺ 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.