Two-photon calcium imaging of the medial prefrontal cortex and hippocampus without cortical invasion
- Graduate School of Medicine, The University of Tokyo, Japan
- National Institute for Basic Biology, Japan
- National Institute for Physiological Sciences, Japan
- Graduate School of Science and Engineering, Saitama University, Japan
Figures
Figure 1
Two-photon imaging with underfilled and overfilled objectives.
(A) Schematic illustration of an objective with NA of 1.0, magnification of 25, and working distance of 4 mm, which was underfilled (left) and overfilled (right) with an excitation laser. …
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Figure 1—source data 1
Data of bright fluorescent signals and the number of detected circles at all depths in four fields in underfilled and overfilled configurations.
Data in Field four were used for top panels in Figure 1D,F.
- https://doi.org/10.7554/eLife.26839.004
Figure 2
Two-photon calcium imaging of intact medial frontal cortex.
(A) Schematic illustration of in vivo two-photon imaging of the mFrC. The dotted square indicates the location at which the Z-stack images in (B) were acquired. Imaging stage was tilted so that the …
Figure 3 with 3 supplements
Immunoreactivity after deep imaging of the medial frontal cortex.
(A–C) Top, representative expression of GFAP (A), Iba1 (B), and HSP70/72 (C) in contralateral (left) and imaged (right) hemispheres. Scale bar, 500 µm. Middle and bottom, expanded images of the …
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Figure 3—source data 1
Data of immunoreactivity across five conditions and their statistics.
All data and p-values for Figure 3D–F are shown.
- https://doi.org/10.7554/eLife.26839.012
Figure 3—figure supplement 1
Immunoreactivity in the negative control experiment.
(A–C) Top, representative expression of GFAP (A), Iba1 (B), and HSP70/72 (c) in contra lateral (left) and AAV-injected and cranial window-set (right) hemispheres. Scale bar, 200 µm. Middle and …
Figure 3—figure supplement 2
Immunoreactivity in the strong positive control experiment.
(A–C) Representative expressions of GFAP (A), Iba1 (B), and HSP70/72 (C) in contralateral (left) and imaged (right) hemispheres (30 min imaging at 300 µm depth was conducted with a 200 mW, 920 nm …
Figure 3—figure supplement 3
Calcium transients of neurons in the deep area before and after 15 min imaging.
(A) Representative calcium-transient traces from neurons at a depth of 1040 µm during the first 5-min period and the last 5-min period of the total 25-min imaging. The same number indicates the same …
Figure 4 with 4 supplements
Neural activity in the medial frontal cortex during conditioning.
(A) Left, representative traces of licking behavior during conditioning sessions 1 (top) and 4 (bottom) from the same mouse. Lines indicate the timing of licking detected by cut-off of light to an …
Figure 4—figure supplement 1
Representative trial-to-trial traces of relative fluorescence change (ΔF/F) for six neurons, aligned to water delivery.
Left, traces from neurons with peak activity during the pre-reward period in the superficial (top), middle (middle), and deep (bottom) areas. Right, traces from neurons with peak activity 0–5 s …
Figure 4—figure supplement 2
Ridge-to-background ratios of the mFrC neurons showing peak activity during the pre-reward period.
Distribution and mean of the ridge-to-background ratios in original and shuffled data from neurons with peak activity during the pre-reward period. Left to right panels correspond to the superficial …
Figure 4—figure supplement 3
Ridge-to-background ratios of neurons showing peak activity during 5 s windows in the pre-reward period.
(A, B) Examples of ridge-to-background ratios calculated from neurons with peak mean activity occurring within six 5 s time windows (from left to right: −10 s to −5 s, −9 s to −4 s, −8 s to −3 s, −7 …
Figure 4—figure supplement 4
Stability of trial-by-trial activity of the mFrC neurons with peak activity during the pre-reward period.
(A) Mean activity of mFrC neurons around the water delivery (dashed lines) from two representative separated groups of trials (top and bottom). In each column, neurons were ordered by the time of …
Figure 5 with 1 supplement
Neural activity in the hippocampal CA1 region during conditioning.
(A) Schematic illustration of in vivo two-photon imaging of the hippocampal CA1 region. The dotted square indicates the location where the Z-stack images in (B) were acquired. Imaging stage was …
Figure 5—figure supplement 1
Expanded immunoreactivity images of the hippocampus after imaging.
(A–C) Top, expression of GFAP (A), Iba1 (B), and HSP70/72 (C) in contralateral (left) and imaged hemispheres. The images were taken from the same mouse imaged in Figure 5C. The tissue was fixed …
Figure 6
Neural activity in the hippocampal CA1 region during conditioning.
(A) Left, mean licking frequency in sessions 1 (red) and 3 (blue; n = 3 mice). Light shading indicates the s.e.m. Middle, response rate in conditioning sessions 1–3 before imaging started (n = 3 at …
Videos
Video 1
Representative two-photon XYZ images of the mFrC in a tdTomato-expressing animal with underfilled and overfilled objectives.
The depth increment in the image stack was 2.5 µm, and the lowest imaging depth was 1120 µm from the cortical surface. The field of view is 509.12 µm × 509.12 µm (512 × 512 pixels). Each image is …
Video 2
Representative two-photon XYZ images of the mFrC expressing R-CaMP1.07.
The depth increment in the image stack was 2.0 µm, and the lowest imaging depth was 1100 µm. The field of view is 509.12 µm × 509.12 µm (512 × 512 pixels). Each image represents an average of 16 …
Video 3
Functional imaging of the PL area expressing R-CaMP1.07 during conditioning.
The imaging depth was 1100 µm from the cortical surface. The field of view is 509.12 µm × 509.12 µm (512 × 512 pixels). White circles at the bottom right indicate the timing of water delivery, with …
Video 4
Representative two-photon XYZ images of neocortex and hippocampus expressing jRGECO1a.
The depth increment in the image stack was 2.5 µm and the lowest imaging depth was 1100 µm. The field of view is the same as in Video 2. Each image is an average of 16 frames. The mouse was not …
Video 5
Functional imaging of the hippocampal CA1 pyramidal layer expressing jRGECO1a during conditioning.
The imaging depth was 1000 µm from the cortical surface and the upper cortical tissue was intact. The field of view is 509.12 µm × 159.04 µm (512 × 160 pixels). White circles at the bottom right …
Additional files
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Transparent reporting form
- https://doi.org/10.7554/eLife.26839.024
