The main aim of GRACE project, in vivo dual simultaneous recording of two-photon imaging and LFP signals in the mouse barrel cortex, was achieved at OIST (Japan) and in UNIPD (Italy) using both viral expressed Calcium indicators and Voltage Sensitive Dyes along with implantable neuro probes. A commercial sensor having a linear 32-channels recording array on the tip was inserted in the mouse brain under the objective of the two-photon imaging setup and LFP signals were recorded simultaneously with imaging data, both during anaesthesia and awake state, while the mouse was free to walk, run or rest on a custom-made treadmill. Once inserted in the brain, the needle of the probe covered 4 out of 6 cortical layers.
Along with simultaneous LFP recordings, two-photon calcium or VSD imaging was acquired at 3 depths, corresponding to layer I, II and IV of the mouse barrel cortex via full-frame scans or box scans (having a much higher imaging frequency).
With this system, both spontaneous and evoked activity could be acquired: during the stimulation epochs, the whiskers of the mouse were deflected by means of air-puffs delivered with random timing, with an average interstimulus interval of about 10 seconds.
Methods for analysing the signals with custom MATLAB and ImageJ routines were designed and implemented during the whole data acquisition period, in parallel with data collection. These algorithms were used to load raw data, remove motion artefacts (frequently generated by the breathing or running of the animal), select Regions of Interest (ROIs) and compute stimulus-locked averages of evoked responses and frequency spectra of Ca2+, VSD and LFP signals. A preliminary analysis was first focused on comparing the shapes of evoked responses in LFP and VSD signals recorded from different cortical layers and different mice and on the frequency content of spontaneous activity traces recorded by both the acquisition systems at the same cortical depths, both during anaesthesia and awake state.
A methodological paper about the new technique and the first results was published in Frontiers in Neuroscience – Brain Imaging Methods with the title "Simultaneous two-photon voltage or calcium imaging and multi-channel LFP recordings in barrel cortex of awake and anesthetized mice" (Authors: C. Cecchetto*, S. Vassanelli, B. Kuhn*,
https://doi.org/10.3389/fnins.2021.741279(se abrirá en una nueva ventana) Associated data available on Dryad at
https://doi.org/10.5061/dryad.dbrv15f23(se abrirá en una nueva ventana)).
