[PhD Thesis Presentation] ‐ Mr. Leonidas Georgiou – “Intercellular AAV transfer from axons to astrocytes allows high contrast activity mapping of astrocytes in awake mice”


Wednesday, February 26, 2020 - 09:00 to 10:00


C210, Ctr Bldg, Level C


Presenter: Mr. Leonidas Georgiou

Supervisor: Dr. Bernd Kuhn

Unit: Optical Neuroimaging Unit

Title: Intercellular AAV transfer from axons to astrocytes allows high contrast activity mapping of astrocytes in awake mice


Astrocytes are more and more considered to be actively involved in information processing in the brain. This idea was primarily established through in vitro experiments. A series of controversies challenged these findings and highlighted the importance of studying astrocytes embedded in their physiological environment that is in awake animals. Astrocytes extend highly ramified processes that form functionally isolated microdomains where they exhibit a rich repertoire of localised calcium signals. How astrocyte microdomain [Ca2+]i signals relate to neuronal activity and behaviour is still unclear. My objective was to investigate circuit specific, single-astrocyte [Ca2+]i microdomain activity in mice during behavioural states and sensory stimuli.
We developed a method for sparse, high contrast labelling of astrocytes embedded in the thalamocortical circuit. We use this technique in combination with two-photon microscopy and genetically encoded calcium indicators (GECIs) in awake mice to study astrocyte [Ca2+]i signals during rest, running, whisker stimulation and thalamocortical axon activity.
Tracking AAV capsids with immunohistochemistry revealed that AAVs injected in the thalamus transfer to astrocytes and neurons in the cortex. This suggests anterograde intercellular transfer of AAVs via thalamocortical axons to cortical astrocytes and neurons. This AAV transfer can be used for the expression of a wide range of genetic tools.
Due to the low probability of AAV transfer we were able to sparsely label astrocytes with membrane tagged GCaPM6f. In combination with two-photon microscopy in awake head fixed mice free to run on a cylindrical treadmill with incorporated whisker stimuli we were able to record single-astrocyte microdomain [Ca2+]i activity. Unbiased analysis of these signals with automated, event based detection software revealed that [Ca2+]i signals in cortical astrocytes exhibit distinct feature changes across behaviour states (run, rest) but not during whisker stimulation.
By exploiting rAAV transfer properties we labelled thalamocortical axons and cortical astrocytes with GECIs. We investigated whether axon bouton calcium signals within the territory of astrocytes correlate with local astrocyte microdomain signals under physiological conditions during different behavioural states. We found no correlation between the calcium activity of boutons and nearby astrocyte microdomains reinforcing our previous observations, suggesting that neurons and astrocytes do not communicate with each other at fast time scales (1.5s).
Finally, we tested if astrocyte microdomain [Ca2+]i signals are random or if astrocytic maps or hotspots exist. Long recordings of single-astrocyte microdomain activity compared to random simulations revealed that astrocytes exhibit regions of higher activity represented as heatmaps. This suggests that astrocyte microdomain [Ca2+]i activity is non-random. Comparing the activity heatmap of the same astrocytes over days revealed that the heatmaps are stable suggesting the existence of astrocyte activity maps in the brain.

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