KAUST Research Workshop on Innovative Technologies to Study Brain Energy Metabolism
University of Lausanne, Switzerland
Professor Volterra holds a PhD in Pharmacology and performed his postdoctoral research at Columbia University in the labs of S. Siegelbaum and Nobel Prize winner, E. Kandel, He then started his independent career in the Dept. of Pharmacology University of Milan and after he moved to the DNF which he directed from 2004 to 2012. Throughout his career, he won several prizes, including the Theodore Ott Prize for Neurosciences in 2017, obtained >30 grants, is author of > 100 publications with >8500 citations and h-index of 38. He has significantly contributed to the seminal work of the astrocyte-neuron communication field in the last twenty years.
Astrocytes sense neuronal inputs and in turn modulate synapses and blood vessels via intracellular Ca2+ signaling. Despite their importance, the properties of astrocytic Ca2+ signals are not well understood. Most of the astrocytic structure presents functional barriers to intracellular Ca2+ propagation, with the result that Ca2+ events are highly compartmentalized throughout the cell, and exhibit large variability in amplitude and duration, as well as spatial spread. It is difficult to reliably study Ca2+ events because only an absolute minority, at best ~5%, of the total astrocyte lies within a given 2-photon focal plane. We report here our recent success in performing fast 3D volumetric imaging, monitoring entire individual astrocytes in adult hippocampal and cortical slices, and in vivo. Morphology of individual astrocytes was visualized using SR101 dye uptake, while genetically-encoded indicator GCaMP6f was used to monitor Ca2+ elevations. Both optically resolved (core) structure of the astrocyte, and optically sub-resolved fine processes (gliapil) were analyzed using two independent strategies. By visualizing the whole astrocyte, we were able to correctly capture and describe the full gamut of Ca2+ events and their properties, including duration, kinetics, and spread volume. The majority of the observed events were three-dimensional, and would not be otherwise properly captured in 2D. Moreover, we were able to simultaneously compare activity across all individual processes on the same cell: something previously impossible due to the limitations of 2D microscopy. We found that most of the activity in the astrocyte core lies outside the soma, and is concentrated in the processes and endfeet. A large portion of this activity is highly compartmentalized and asynchronous. As a further practical application of the 3D method, we were able to study functional interactions between astrocytes and axons, and between endfeet and blood vessels, as well as astrocytic population activity in vivo. Our volumetric imaging and analysis method has important implications outside the glial Ca2+ field. While independently surveilling tens of thousands of μm3 per cell, we identified the presence of multiple “hot” and “cold spots,” and observed a large diversity of highly compartmentalized Ca2+ signals happening within a single astrocyte, as well as functional “coupling” between some but not other “voxels”. This is methodologically comparable to monitoring and analyzing the connections between thousands of neuronal cell bodies within a brain circuit/network. We believe our methods and strategies will therefore be useful also for neuronal network Ca2+ analysis. This work was supported by grants: ERC Advanced "Astromnesis" and SNSF 31003A-173124 to AV.