Fig 1.
Transgene expression in TRE-G-CaMP7 mice crossed with CaMKII-tTA driver mice.
A, Design and strategy for controlled G-CaMP7 expression in TRE-G-CaMP7 mice. G-CaMP7 expression is controlled spatially and temporally by a cell-type-specific tTA driver mouse line and doxycycline (Dox) administration, respectively. PCaMKIIα, CaMKIIα promoter; PminCMV, minimal cytomegalovirus promoter. B, Expression patterns of G-CaMP7 and DsRed2 in 1-month-old mice. Scale bar = 2 mm. C, Expression of G-CaMP7 and DsRed2 in layer 2/3 (L2/3) and layer 5 (L5) pyramidal neurons in the visual cortex and in CA1 pyramidal neurons in the hippocampus. Scale bar = 20 μm. D, Expression of G-CaMP7 in CaMKIIα-positive pyramidal neurons in layer 2/3 of the visual cortex. Scale bar = 20 μm. E, Reversible control of transgene expression by Dox administration. Parasagittal sections were cut from mice without Dox administration (Dox (-)), after 2 weeks of Dox administration (Dox (+)), and after 2 weeks of Dox withdrawal (Dox (+)→(-)). All the sections were cut from mice at 4–5 months of age, and the images were obtained with the same exposure time. Scale bar = 2 mm.
Fig 2.
Transgene expression in brain areas outside the neocortex and hippocampus.
The expression patterns of G-CaMP7 and DsRed2 in coronal sections of the olfactory bulb, piriform cortex, amygdala and striatum from TRE-G-CaMP7 x CaMKII-tTA mice are shown at lower (left panels for each brain area) and higher (right panels) magnifications. Arrowheads indicate olfactory glomeruli intensely labeled by G-CaMP7. Arrows represent examples of striatal neurons strongly expressing G-CaMP7. Gl, glomerular layer; EP, external plexiform layer; MC, mitral cell layer; IP, internal plexiform layer; Gr, granule cell layer; I, II, and III, layers I, II, and III of the piriform cortex, respectively; LA, lateral amygdala; BLA, basolateral amygdala; St, striatum; EC, external capsule; D, dorsal; L, lateral; M, medial. Scale bar = 100 μm.
Fig 3.
Two-photon imaging of spontaneous cortical layer 2/3 circuit activity in anesthetized TRE-G-CaMP7 x CaMKII-tTA mice at 4 months of age.
A, Neurons expressing G-CaMP7 (left) and DsRed2 (right) were imaged in the posterior cortex 60, 200, and 350 μm from the pial surface. The darker areas in each image are shadows of vessels. Scale bar = 100 μm. B, The positions and numbers of 13 active neurons are indicated in an average G-CaMP7 fluorescence image acquired at a depth of 200 μm (left). Similarly, those of 6 neuropil areas are shown in the same average G-CaMP7 fluorescence image (right). C, Baseline-normalized G-CaMP7 fluorescence time traces for the same 13 cells (top) and 6 neuropil regions (bottom left) and baseline-normalized DsRed2 fluorescence time traces for the same 6 neuropil regions (bottom right). D, A cross-correlation matrix of G-CaMP7 fluorescence time traces of the 13 cells. E, Example time-lapse images of G-CaMP7 fluorescence during spontaneous network activity in layer 2/3 of the cortex. Cell 2 and Cell 13 (as designated in B) were synchronously activated multiple times. Time stamps are indicated above the images. Active cells are indicated by red arrows.
Fig 4.
Two-photon imaging of spontaneous hippocampal CA1 circuit activity in anesthetized TRE-G-CaMP7 x CaMKII-tTA mice at 4 months of age.
A, Different subcellular compartments of pyramidal neurons expressing G-CaMP7 (left) and DsRed2 (right) were imaged in the dorsal CA1 hippocampus 50, 95 and 140 μm from the hippocampal surface. Scale bar = 50 μm. B, The positions and numbers of 12 active neurons selected for analysis are indicated in an average G-CaMP7 fluorescence image acquired at a depth of 145 μm. C, Baseline-normalized G-CaMP7 fluorescence time traces of the 12 active cells. D, A cross-correlation matrix of G-CaMP7 fluorescence time traces of the 12 active cells. E, Example time-lapse images of G-CaMP7 fluorescence during spontaneous network activity in the CA1 hippocampus. Cells 7 and 2 (as designated in B) were sequentially activated multiple times. Only two out of nine total sequential activation events are shown here. Time stamps are indicated at the bottom of the images. Δt represents the image sampling interval (0.43 s). Active cells 7 and 2 are indicated by red arrows.