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Deep two-photon brain imaging with a red-shifted fluorometric Ca2+ indicator - PubMed

  • ️Thu Jan 01 2015

Deep two-photon brain imaging with a red-shifted fluorometric Ca2+ indicator

Carsten Tischbirek et al. Proc Natl Acad Sci U S A. 2015.

Abstract

In vivo Ca2+ imaging of neuronal populations in deep cortical layers has remained a major challenge, as the recording depth of two-photon microscopy is limited because of the scattering and absorption of photons in brain tissue. A possible strategy to increase the imaging depth is the use of red-shifted fluorescent dyes, as scattering of photons is reduced at long wavelengths. Here, we tested the red-shifted fluorescent Ca2+ indicator Cal-590 for deep tissue experiments in the mouse cortex in vivo. In experiments involving bulk loading of neurons with the acetoxymethyl (AM) ester version of Cal-590, combined two-photon imaging and cell-attached recordings revealed that, despite the relatively low affinity of Cal-590 for Ca2+ (Kd=561 nM), single-action potential-evoked Ca2+ transients were discernable in most neurons with a good signal-to-noise ratio. Action potential-dependent Ca2+ transients were recorded in neurons of all six layers of the cortex at depths of up to -900 µm below the pial surface. We demonstrate that Cal-590 is also suited for multicolor functional imaging experiments in combination with other Ca2+ indicators. Ca2+ transients in the dendrites of an individual Oregon green 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-1 (OGB-1)-labeled neuron and the surrounding population of Cal-590-labeled cells were recorded simultaneously on two spectrally separated detection channels. We conclude that the red-shifted Ca2+ indicator Cal-590 is well suited for in vivo two-photon Ca2+ imaging experiments in all layers of mouse cortex. In combination with spectrally different Ca2+ indicators, such as OGB-1, Cal-590 can be readily used for simultaneous multicolor functional imaging experiments.

Keywords: calcium imaging; mouse cortical circuits; multicolor functional imaging; neuronal activity.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.

Cal-590-based in vivo two-photon Ca2+ imaging in bulk-loaded cortical layer 2/3 and 4 neurons. (A) Scheme of two-photon microscope with optimized components for long-wavelength measurements in vivo. PMT, photomultiplier; RS, resonant scanner. (B) Cal-590 two-photon excitation spectrum measured with 0 and 2 mM Ca2+. (C) In vivo two-photon image (Left, average of 30-s recording time) of the focal plane of cortical layer 2/3 neurons used to measure the Ca2+ transients (Right) in the cell indicated by the red dotted line. Action potentials (APs), numbers indicated above the traces, were recorded in the cell-attached mode (bottom gray trace). (D) Averaged Ca2+ transient in response to a single AP (mean of 54 events in three animals). Red dotted line indicates single exponential fit. (E) Two-photon image (reconstruction from z-stack) representing the side view of the visual cortex labeled in layer 4 with Cal-590 AM. Depth values as indicated. (F) Image of the focal plane (−491 µm; see red arrow in E) used for the recording of spontaneous Ca2+ transients. Traces (Right) correspond to the cells indicated in the image.

Fig. S1.
Fig. S1.

Action potential-evoked neuronal Ca2+ transients after staining with Cal-590 AM and OGB-1:00 AM, respectively. (A) Example of combined two-photon Ca2+ imaging (top traces) and cell-attached recordings (bottom traces) in a layer 2/3 neuron of the visual cortex in an anesthetized mouse. Recordings were obtained after bulk-loading with Cal-590 AM. Numbers below the traces indicate the number of action potentials. (B) Similar recordings obtained after bulk-loading with OGB-1:00 AM in another mouse. Recordings in A and B were obtained in the same set-up and under identical recording conditions.

Fig. S2.
Fig. S2.

Staining of astrocytes with Cal-590 in the in vivo mouse cortex. (A) Two-photon fluorescence image (average of 1,200 consecutive frames) obtained in layer 2/3 of the visual cortex of an anesthetized mouse after bulk-loading with Cal-590 AM. The excitation wavelength was 1,050 nm. (B) Two-photon fluorescence image of the same optical section as in A after additional staining with sulforhodamine 101 (SR 101). For staining with SR 101, we used the protocol initially published by Nimmerjahn et al. (19). The excitation wavelength was 900 nm. The red arrows here and in A indicate two labeled astrocytes.

Fig. 2.
Fig. 2.

Cal-590-based imaging in electroporated single layer 5 neurons. (A) Morphology of a pyramidal neuron in layer 5 of the mouse visual cortex that was labeled in vivo via Cal-590 electroporation and reconstructed from a z-stack projection. (Insets) Two-photon images of the focal planes used to image the dendritic trunk and the soma at depths, as indicated. White dotted lines indicate the ROIs used for data analysis. (B) Ca2+ transients associated with spontaneous action potential (AP) activity recorded in the apical dendritic trunk and in the soma at depths of −500 and −597 µm, respectively, in combination with cell-attached recordings (bottom gray traces). (C) Dendritic Ca2+ transient in response to a single AP (mean of 14 events recorded in the dendritic trunk indicated in A). Red dotted line indicates single exponential fit. (D) Rising phase of a Ca2+ transient associated with a train of three APs (bottom gray trace). Imaging performed at 500 frames/s in the dendritic trunk segment shown in A. (E) Graph of the number of APs vs. dendritic and somatic Ca2+ transient amplitudes (mean ± SD). Red traces represent the corresponding linear fits. (Inset) Overlay of dendritic Ca2+ signals corresponding to one, two, and three APs, respectively.

Fig. 3.
Fig. 3.

Cal-590-based two-photon Ca2+ imaging in deep cortical layers 5 and 6. (A) Two-photon image (reconstruction from z-stack) representing the side view of the visual cortex labeled in layer 5 with Cal-590 AM. Depths values as indicated. (B) Image of the focal plane (−665 µm, see red arrow in A) used for the recording of spontaneous Ca2+ transients. Traces (Bottom) correspond to the cells indicated in the image. (C and D) Two-photon imaging of layer 6 neurons (depth, −870 µm). Similar arrangement as that shown in A and B for layer 5. (E) Two-photon image of a Cal-590 AM-stained layer 5 neuron with the recording patch-pipette (Left). Traces of AP activity (bottom) and the corresponding Ca2+ transients (Top). Numbers of APs as indicated. (F) Graph of the number of APs vs. Ca2+ transient amplitudes (mean ± SD). Red trace represents the corresponding linear fit.

Fig. 4.
Fig. 4.

Dual-channel and two-color hybrid functional Ca2+ imaging. (A) Two-photon image (depth, −500 µm; 1,050-nm excitation wavelength) of deep cortical layer 4 neurons stained with Cal-590 AM (Top). Spontaneous Ca2+ transients recorded in the ROIs corresponding to two neurons (dotted circles). The arrow indicates the neuron selected for OGB-1 electroporation. (B) Two-photon image of the same region as in A (but with 920-nm excitation wavelength) depicting a neuron electroporated with OGB-1 and its dendrites (image of dendrites obtained from an average projection of five focal planes, recorded 10 µm apart). (Inset) Two spiny dendrites, magnification of the image shown in the white rectangle. Spontaneous Ca2+ transients in the two dendrites were recorded simultaneously in the corresponding dendrites, as indicated. (C) Overlay of a two-photon image of Cal-590 AM-labeled layer 2/3 neurons (excitation wavelength, 1,000 nm; emission light collected between 590 and 650 nm, red channel) and a simultaneously imaged neuron (electroporated with OGB-1; excitation wavelength, 1,000 nm; emission light collected between 425 and 550 nm, green channel). (D and E) Separate display of images from the red and green channels representing the dotted rectangular region indicated in C. (F) Magnification of the dotted rectangular region indicated in C. (G) Simultaneously recorded Ca2+ transients from three Cal-590-labeled neurons (red channel) and from a dendritic segment of the OGB-1-labeled neuron (green channel), as indicated in F.

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