Abstract
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.
Highlights
Ca2+-dependent, stochastic release of glutamate quanta is a fundamental function of excitatory synapses [1, 2]
We present an all-optical approach to simultaneous monitoring of Ca2+ dynamics within two segmented compartments of the tripartite synapse in response to a physiological stimulus
The technique involves the employment of a multiplexed two-photon microscopy system capable of efficiently exciting spectrally distinct, genetically-encoded optical sensors, which are strategically targeted to various regions of the brain
Summary
Ca2+-dependent, stochastic release of glutamate quanta is a fundamental function of excitatory synapses [1, 2]. The presence of nanoscopic extensions of astrocytes that surround many synapses and demonstrate activity-locked Ca2+ excitability has hastened our reevaluation of astrocytic functions and their role in neurotransmission and signal integration in the brain [3,4,5]. The copyright holder for this preprint It is made available under. Thalamocortical projections of the ventrobasal complex (VB) in the thalamus are an ideal target for the selective and sparse labelling of presynaptic elements within the cortex. We demonstrate that labelling of axonal boutons in this way can be coupled with labelling of cortical astrocytes, to reliably yield simultaneous readouts of perisynaptic (astrocytes) and presynaptic (thalamocortical-projecting neurons) Ca2+ fluctuations during evoked and spontaneous cortical activity at single synapses. A more exhaustive study of such dynamics might yield important insight into the contribution of astrocytes to different thalamus-mediated behaviours, and the heterogeneity of such contributions [6]
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
