Domonkos Pinke1, Máté Marosi1, Gergely Dobos2, Csaba Csupernyák1, Dominika Nagy1, Gergely Szalay1, Andrius Plauska1, Gergely Katona1, Balázs Rózsa1
1 Institute of Experimental Medicine of Hungarian Academy of Sciences, Budapest
2 Bay Zoltán Foundation for Applied Research, Budapest
The systematic understanding of brain function requires methods that allow neuronal activity to be recorded at different spatial scales in 3D at a high temporal resolution. Recording techniques are required that collect information from a neuronal population situated in an extensive volume of tissue.
Aims: Technical challenge of two-photon imaging in long-term is the stability of cell recordings. Lateral and Z displacement can be managed with 2D shift scanning and Z-focusing, however, effects of XYZ rotation and tissue deformation remain unsolved.
Here we present a novel fast 3D volumetric imaging method, suitable for long-term in vivo tracking of neuronal activity in mouse cortex by acusto-optical two-photon microscopy. We could precisely identify and record from the center plane of cell bodies and track the visually evoked neuronal activity. On the first day of the experiment after a control Z-stack acquisition in near-cubic-millimeter scan range, 3D drifting acusto-optical volumetric imaging (50x50x50µm Multi-cube scans) were recorded around the selected cell bodies. On the next imaging sessions the recording coordinates were re-loaded to the acquisition software and the recording sites were roughly identified by the vascular architecture. Then new Multi-cube scans were acquired and used for fine alignment in a 3D volume.
With the Multi-cube scanning method we are able to locate and record Ca2+ activity (with GCaMP6f) of the same neuronal ensemble during our 10-20 days long protocol including baseline, training and post learning imaging sessions of up to 200 cells. Our results show this method is significantly more accurate than a conventional Z-stack ROI selection.
We demonstrate a method, that allows us to record long term neuronal plasticity from the same cell population in a near-cubic millimeter 3D volume, up to 2 months. For this reason, the Multi-cube scanning method is suitable for long term imaging behavior protocols.
Doctoral School: Szentágothai János Doctoral School of Neuroscience
Program: Functional Neuroscience
Supervisor: Balázs Rózsa
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