Multiplexed 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.\n\nHighlightsO_LIWe applied multiplexed multiphoton imaging to optically assess activity of individual synapses and the surrounding astroglia in the intact brain\nC_LIO_LIPerisynaptic astrocytic regions display localiased, context-dependent Ca2+ elevations in vivo\nC_LIO_LISuch elevations may precede spontaneous Ca2+ entry at presynaptic boutons\nC_LIO_LIThis method paves the way for optical investigation of glutamate release probability in vivo\nC_LI