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Simultaneous two-photon voltage or calcium imaging and multi-channel LFP recordings in barrel cortex of awake and anesthetized mice


Cecchetto, Claudia; Vassanelli, Stefano; Kuhn, Bernd (2021), Simultaneous two-photon voltage or calcium imaging and multi-channel LFP recordings in barrel cortex of awake and anesthetized mice, Dryad, Dataset,


Neuronal population activity, both spontaneous and sensory-evoked, generates propagating waves in cortex. However, high spatiotemporal-resolution mapping of these waves is difficult as calcium imaging, the work horse of current imaging, does not reveal subthreshold activity.

Here, we present a platform combining voltage or calcium two-photon imaging with multi-channel local field potential (LFP) recordings in different layers of the barrel cortex from anesthetized and awake head-restrained mice. A chronic cranial window with access port allows injecting a viral vector expressing GCaMP6f or the voltage-sensitive dye (VSD) ANNINE-6plus, as well as entering the brain with a multi-channel neural probe. We present both average spontaneous activity and average evoked signals in response to multi-whisker air-puff stimulations.

Time domain analysis shows the dependence of the evoked responses on the cortical layer and on the state of the animal, here separated into anesthetized, awake but resting, and running. The simultaneous data acquisition allows to compare the average membrane depolarization measured with ANNINE-6plus with the amplitude and shape of the LFP recordings. The calcium imaging data connects these data sets to the large existing database of this important second messenger. Interestingly, in the calcium imaging data, we found a few cells which showed a decrease in calcium concentration in response to vibrissa stimulation in awake mice.

This system offers a multimodal technique to study the spatiotemporal dynamics of neuronal signals through a 3D architecture in vivo. It will provide novel insights on sensory coding, closing the gap between electrical and optical recordings.


Chronic cranial surgery

Chronic cranial window surgeries (for detailed protocol see (Augustinaite and Kuhn, 2020a)) were performed on male C57/BL6 mice, 67- to 129-days old using a 5 mm glass cover slip with off-center silicone access ports as described previously (Roome and Kuhn, 2014, 2019). For ECoG recordings, one small hole was drilled into the skull on both hemispheres and in each a silver wire was inserted between the skull and the dura and glued with superglue.

Injection through the access port

For voltage imaging experiments an ANNINE-6plus labeling solution was prepared (for detail protocol see (Roome and Kuhn, 2019)): a stock solution of ANNINE-6plus was prepared in 20% Pluronic F-127 in DMSO at a concentration of 2.0 mM. This stock solution was kept up to a year at room temperature protected from light. Before use, the stock solution was heated to 70°C in a heating block for at least 30 minutes. The heated ANNINE-6plus stock solution was then diluted to 5% in saline (0.9% NaCl in H2O) and immediately filled into the injection pipette.

After a few days of recovery, for calcium imaging experiments, an AAV for GCaMP6f expression (AAV1.Syn.GCaMP6f.WPRE.SV40, addgene) or, for voltage imaging experiments, ANNINE-6plus were injected through the silicone access port at the two-photon microscope setup, using a 20 to 30-degree beveled glass pipette with an opening between 5 and 10 µm. The pipette entered the port at an angle of 27°. AAV was injected 300 µm and 600 µm below the dura, and ANNINE-6plus was injected 400 µm below the dura. AAV was injected at a rate of 10 nl/min, with a total amount of 140 nl for each injection depth. ANNINE-6plus delivery was performed slowly and carefully, aiming for about 700 nl of dye solution being delivered over 1 hour.


We used a custom-built combined wide-field and two photon microscope (MOM, Sutter) with a 5x/N.A. 0.13 air objective (Zeiss) or a 25x/N.A. 1.05 water immersion objective with 2 mm working distance (Olympus). A femtosecond-pulsed Ti:sapphire laser was used to excite fluorescence which was detected by two GaAsP photomultiplier tubes (Hamamatsu) in the spectral range of 490-550 nm (green) and 550-750 nm (red), separated by a dichroic mirror at 552 nm (all Semrock). The microscope was controlled by commercial software (MScan, Sutter Instruments).

Calcium imaging was recorded with 512x512 pixels full-frame scans. The field of view was 375x375 µm2 and the sampling frequency 30.9 Hz. For voltage imaging, boxscans with 512x32 pixels (375x24 µm2, 500 Hz) and 512x16 pixels (375x12 µm2, 1000 Hz) were acquired. For every imaging depth and boxscan type, 10 minutes long scans were acquired. One epoch was taken with no stimulations (i.e., spontaneous activity), while the following was taken while multi-whisker air-puff stimuli (100 ms, 20 psi) were delivered to the contralateral whisker pad with random timing and average interstimulus interval of 10 seconds. Images were acquired both during light anesthesia (1% isoflurane) and wakefulness (resting or running). At the beginning of the experiment and right after probe insertion, z-stacks (total travel of 540 µm with 2 µm z-steps, 50 averages per plane) were acquired. During anesthesia, the mouse body temperature was monitored and kept at 37 °C by means of a heating pad equipped with a rectal probe. Awake recordings followed, after recovery from anesthesia for 30-40 mins. During wakefulness, movements of the cylindrical treadmill were recorded through a rotary encoder (E6A2-CW3C, OMRON).


Electrophysiological signals were acquired by a silicon-based probe with a linear matrix of 32 electrodes (ATLAS Neuro Probe: E32+R-50-S1-L10 NT; pointy tip feature; IrOx electrodes; spacing between electrodes: 50 µm) and connected via an SPI cable to the acquisition system (Open Ephys, OEps Tech). The acquisition board was equipped with an I/O board for interfacing with auxiliary devices: the air-puff TTL and the frame sync signal from the two-photon microscope were acquired and used to synchronize the electrophysiological signal with the stimulation and the image acquisition. While the mouse was anesthetized, the ATLAS probe was inserted into the tissue at a depth of 850 µm under an angle of 27° to the cranial window under visual control using an sCMOS camera (PCO.edge 4.2, PCO) under white light illumination. The probe was inserted using a micromanipulator (Sutter MP-285) at slow speed (30-50 µm/s). Particular attention was given to inserting the probe with the side containing the array facing downwards, i.e. away from the excitation laser beam, to minimize the amount of light impinging on the electrodes. The LFP signal was visualized, recorded, and digitalized at 10 kHz through an open-source software interface supplied with the acquisition system.

The electrocorticogram (ECoG) was recorded at 1 kHz through the silver wires implanted during the surgery using an EEG preamplifier (100× gain, sigmann elektronik GmbH) with additional band-pass filtering (0.5 – 200 Hz; Model 440, Brownlee precision).

Data analysis

Movies were analyzed with custom MATLAB code and ImageJ. ROI selection from calcium imaging data was done in ImageJ. Image registration and motion correction were done in MATLAB using a fast variational method (Flotho et al., 2021).

Electrophysiological signals were converted from .continuous filetype (Open Ephys output files) to .mat files and analyzed through custom MATLAB routines.

Signals acquired by the imaging and electrophysiology setups were synchronized based on the frame sync and TTL signals.

Usage Notes

Data are organized according to the figures in the manuscript.

Usage notes are uploaded in every data folder.


H2020 Marie Skłodowska-Curie Actions, Award: 796177

Okinawa Institute of Science and Technology Graduate University

2018 Canon Foundation Research Fellowship