Simultaneous Two-Photon Voltage or Calcium Imaging and Multi-Channel Local Field Potential Recordings in Barrel Cortex of Awake and Anesthetized Mice

Abstract

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.

Keywords: ANNINE-6; combined approach; cortex; local field potentials; neuroimaging; two-photon microscopy; voltage imaging; whisker stimulation.

FIGURE 1

Scheme of the experimental setup, probe insertion and reconstruction of the insertion track. (A) A mouse is head-fixed on a cylindrical treadmill. An electrical probe (Atlas Neuroengineering) is inserted through the access port of the chronic cranial window into the brain and allows electrical recording. The window allows two-photon imaging of voltage or calcium depth resolved. For sensory stimulation, air puffs can be applied. (B) Mouse cranial window before probe insertion (left), with the tip of the probe positioned on the surface of the silicon plug (center), and with the probe inserted 850 μm deep into cortex (right). (C) ANNINE-6plus fluorescence in a 100-μm thick sagittal brain slice (LM ≈ 2.3 mm). The pipette for loading ANNINE-6plus and the electrical probe are entering through the same port under the same angle. Therefore, the track of the probe insertion is very well approximated by the track of ANNINE-6plus labeling. CTX: cortex, somatosensory areas; VL: lateral ventricle; HPF: hippocampal formation (D) Two-photon reconstruction of cortical neurons expressing GCaMP6f before (left) and after (right) probe insertion with tip below imaging site. Imaging was done at the center of the craniotomy, right below the access port (AP 1.5 mm; LM 2.5 mm). Cortical layers were identified based on (DeFelipe, 2002) and indicated on the left of the image.

FIGURE 2

Simultaneously recorded average VSD signal and LFP, both in barrel cortex, ECoG, and running speed in response to a facial air puff (one mouse). (A) A representative imaged cortical region: 375 × 375 μm² full-frame scan and correspondent 375 × 24 μm² box-scan imaged at 130 μm deep in the cortex. (B–D) VSD responses evoked by an air puff to the vibrissae and cheek of the mouse recorded in 375 × 24 × 5 μm³ and 375 × 12 × 5 μm³ box-scans from a single mouse at three different depths (B) during anesthesia (50 μm: 139 trials, 130 μm: 139 trials, and 350 μm: 138 trials), (C) in awake mouse during resting (50 μm: 127 trials; 130 μm: 125 trials; and 350 μm: 121 trials), and (D) in awake mouse during running (50 μm: 7 trials; 130 μm: 8 trials; and 350 μm: 12 trials). (E–G) LFP recorded in a single mouse by eight electrodes (every 4th channel of the linear probe, corresponding to a distance of 200 μm along the probe, Δdepth = 68 μm between adjacent traces). The probe was inserted with the tip at 850 μm below the dura. Each channel shown is the average of the same number of trials as for the imaging (light blue at 50 μm, blue at 130 μm, and dark blue at 350 μm). The bottom LFP corresponds to the electrode located 800 μm below the dura, the top LFP to the electrode located 100 μm below the dura. Corresponding (H–J) average ECoG and (K–M) average running speed. Dashed gray lines indicate the stimulus onset. All traces are plotted as mean ± standard deviation.

FIGURE 3

Average VSD signal 50 μm below dura and LFP, both in barrel cortex, ECoG, and running speed in response to a facial air puff (six mice). Average VSD responses evoked by an air puff to the vibrissae and cheek of the mouse in layer I (50 μm below dura) recorded in 375 × 24 × 5 μm³ and 375 × 12 × 5 μm³ box-scans in six mice (A) during anesthesia (133, 187, 139, 139, 126, 134 trials), (B) in awake mice during resting (111, 165, 118, 127, 104, 123 trials), and (C) in awake mice during running (16, 27, 14, 7, 16, 11 trials). (D–F) Corresponding average LFP recorded by eight electrodes (every 4th channel of the linear probe, tip at 850 μm below dura, bottom LFP trace: 800 μm, top LFP trace: 100 μm below dura, Δdepth = 68 μm between adjacent traces). Corresponding (G–I) average ECoG and (J–L) average speed. Each mouse is indicated by a different color; the overall average is shown in black. Gray dashed line marks the air puff onset.

FIGURE 4

Average VSD signal 130 μm below dura and LFP, both in barrel cortex, ECoG, and running speed in response to a facial air puff (six mice). Average VSD responses evoked by an air puff to the vibrissae and cheek of the mouse in layer II (130 μm below dura) recorded in 375 × 24 × 5 μm³ and 375 × 12 × 5 μm³ box-scans in six mice (A) during anesthesia (135, 187, 139, 139, 130, 138 trials), (B) in awake mice during resting (109, 195, 125, 125, 96, 124 trials), and (C) in awake mice during running (19, 1, 7, 8, 13, 11 trials). (D–F) Corresponding average LFP recorded by 8 electrodes (every 4^th channel of the linear probe, tip at 850 μm below dura, bottom LFP trace: 800 μm, top LFP trace: 100 μm below dura, Δdepth = 68 μm between adjacent traces). Corresponding (G–I) average ECoG and (J–L) average speed. Each mouse is indicated by a different color; the overall average is shown in black. Gray dashed line marks the air puff onset.

FIGURE 5

Average VSD signal 350 μm below dura and LFP, both in barrel cortex, ECoG, and running speed in response to a facial air puff (six mice). Average VSD responses evoked by an air puff to the vibrissae and cheek of the mouse in layer IV (350 μm below dura) recorded in 375 × 24 × 5 μm³ and 375 × 12 × 5 μm³ box-scans in 6 mice (A) during anesthesia (131, 189, 125, 138, 122, 136 trials), (B) in awake mice during resting (119, 174, 124, 121, 107, 112 trials), and (C) in awake mice during running (16, 5, 8, 12, 2, 22 trials). (D–F) Corresponding average LFP recorded by eight electrodes (every 4th channel of the linear probe, tip at 850 μm below dura, bottom LFP trace: 800 μm, top LFP trace: 100 μm below dura, Δdepth = 68 μm between adjacent traces). Corresponding (G–I) average ECoG and (J–L) average speed. Each mouse is indicated by a different color; the overall average is shown in black. Gray dashed line marks the air puff onset.

FIGURE 6

Average calcium signal at three different depth and LFP, both in barrel cortex, ECoG, and running speed in response to a facial air puff (four anesthetized and awake but resting mice). Average calcium signals recorded from three different depths in (A) anesthetized (4 mice, 50 μm: 236 trials, 55 ROIs, 130 μm: 221 trials, 44 ROIs, 400 μm: 228 trials, 50 ROIs) and awake but resting mice, the latter separated in (B) positive (4 mice, 50 μm: 213 trials, 42 ROIs; 130 μm: 227 trials, 42 ROIs, 400 μm: 215 trials, 38 ROIs) and (C) negative signals (4 mice, 50 μm: 213 trials, 15 ROIs; 130 μm: 227 trials, 20 ROIs, 400 μm: 215 trials, 20 ROIs). (D,E) Corresponding average LFP recorded by eight electrodes (every 4th channel of the linear probe, tip at 850 μm below dura, bottom LFP trace: 800 μm, top LFP trace: 100 μm below dura, Δdepth = 68 μm between adjacent traces). Corresponding (F,G) average ECoG and (H,I) average speed. All traces are plotted as mean ± standard deviation. Gray dashed line marks the air puff onset.

FIGURE 7

Average calcium signal at three different depth and LFP, both in barrel cortex, ECoG, and running speed in response to a facial air puff (four awake mice during running). Average calcium signals recorded from three different depths in awake mice during running, separated in (A) positive (4 mice, 50 μm: 24 trials, 32 ROIs; 130 μm: 9 trials, 37 ROIs; 400 μm: 25 trials, 30 ROIs) and (B) negative signals (4 mice, 50 μm: 24 trials, 24 ROIs, 130 μm: 9 trials, 12 ROIs, 400 μm: 25 trials, 28 ROIs). (C) Corresponding average LFP recorded by eight electrodes (every 4th channel of the linear probe, tip at 850 μm below dura, bottom LFP trace: 800 μm, top LFP trace: 100 μm below dura, Δdepth = 68 μm between adjacent traces. Corresponding (D) average ECoG and (E) average speed. All traces are plotted as mean ± standard deviation. Gray dashed line marks the air puff onset.