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BSTRACTRecent evidences suggest that astrocytes participate actively in the computational potential of the neocortex interacting with neurons at multiple spatio-temporal scales. Indeed astrocyte have been demonstrated regulate basal synaptic transmission and respond along with neurons upon sensory stimulation. A fine dissection of the contribution of different neural classes to the cortical function is necessary to elucidate the nature of these interactions in vivo. I addressed this issue by performing targeted two-photon calcium imaging of neurons and astrocytes and electrophysiology in the mouse primary visual cortex in vivo.
The combination of two-photon in vivo imaging and of the genetic labeling of specific cell types in the mouse brain is a powerful method to refine our understanding of cortical circuitry Unfortunately, the spectral properties of Oregon Green BAPTA1 and EGFP, the most widely used fluorophores for targeted two photon imaging, are overlapped.
First I characterized the in vivo spectral properties EGFP and Oregon Green Bapta 1. Here I present a strategy for spectral un-mixing of fluorescence to identify cells tagged with EGFP and to correct Ca2+ dynamics. In principle, this approach could be extended to others combinations of fluorophore provided that have sufficiently different two-photon excitation spectra. The development of a multi-colour cell tagging scenario combined with Ca2+ dynamic measures could have a dramatic impact on the capabilities of studying neural interactions and cortical fine scale physiology.
Then I sought to investigate how astrocytes cooperate with neurons to process sensory information in the mouse primary visual cortex. I adapted the two photon microscopy setup to monitor V1 astrocytes recruitment with subcellular resolution by means of targeted calcium imaging during presentation of different visual stimuli. Here I report preliminary results describing visual evoked responses in neurons and astrocytes soma and primary processes.
