PMC

Sparse GEVI expression in vivo resolves subcellular structures. (a) At full expression density, GEVI optical signals are difficult to be assigned to individual neurons due to intermingled neuronal processes in dense populations. (b) Sparse Golgi staining-like GEVI expression density resolves single cells and subcellular morphologies (i) A 15 µm stack projection of three individual layer II/III pyramidal neurons. (ii) Single-plane image from the stack in (b-i). GEVI targeting achieves optimal membrane expression, and is capable of resolving subcellular structures in vivo, including axonal boutons (blue inset) and dendritic spines (white inset). Image scale bar = 50 µm; inset scale bar = 2 µm.

Dual-controlled co-expression of GECI (GCaMP6f) and GEVI (VSFP Butterfly 1.2) in somatosensory cortical layer II/III pyramidal neurons in vivo. (a) The combination of dCre and tTA allows for dual control of Ai78 VSFP Butterfly 1.2 and Ai93 GCaMP6f expression probabilities. This transgenic strategy resulted in GECI-expressing, GEVI-expressing and GECI/GEVI co-expressing populations. (b-i) Strong GCaMP6f fluorescence signal observed in perisomatic regions of individual pyramidal neurons. (ii) Strong VSFP Butterfly 1.2 fluorescence signal observed in the membranes of two pyramidal neurons (arrows). (b-iii) Merged GCaMP6f (green) and VSFP Butterfly 1.2 (red) fluorescence signals on DAPI nuclei counterstaining (blue) showing sparse Golgi staining-like indicator expression pattern. Inset: a single-plane confocal image of the indicator-expressing neuron, outlined by the dotted box in (b-ii), showing optimal GEVI membrane targeting. Note this cell co-expresses GCaMP6f and VSFP Butterfly 1.2. Scale bar = 50 µm.

Expression of genetically encoded voltage indicator (chimeric VSFP Butterfly) in all cortical pyramidal neurons with controlled co-expression of genetically encoded calcium indicator (GCaMP6f) in layer II/III pyramidal neurons in vivo. (a) A transactivator, driven by CaMK2A promoter (CaMK2A-tTA) in pyramidal neurons, is required for binding to the TRE promoter for transcription activation in both chimeric VSFP Butterfly and Ai93 indicator mice. A STOP cassette removal using TMP-stabilized Cre-recombinase driven by Rasgrf2-2A promoter (Rasgrf2-2A-dCre) is only needed for the Ai93 line. This quadruple transgenic strategy therefore achieves full GEVI expression in all pyramidal neurons, with TMP dose-dependent adjustable GECI co-expression in layer II/III pyramidal neurons (b-i) Strong GCaMP6f fluorescence signal observed in perisomatic regions of individual pyramidal neurons (b-ii) A single confocal plane image showing strong chimeric VSFP Butterfly fluorescence signal from pyramidal neurons observed across all cortical layers (b-iii) Merged GCaMP6f (green) and chimeric VSFP Butterfly (red) fluorescence signals showing adjustable sparse GECI indicator expression over dense GEVI expression patterns. Scale bar = 50 µm.

Representative images of adjusted GECI expression probability of layer II/III pyramidal neurons achieved via dose-dependent TMP administration. (a) Transgenic strategy using the combination of both Cre-recombinase and transactivator intersectionally controls indicator expression in identified cell populations in Ai93 GCaMP6f indicator transgenic mice. The transgenic strategy shown here uses a destabilized form of Cre-recombinase driven by Rasgrf2-2A promoter (Rasgrf2-2A-dCre) that can be “de-destabilized” using TMP to remove the STOP cassette in layer II/III neurons. Transactivator expression driven by CaMK2A promoter (CaMK2A-tTA) in pyramidal neurons is also required for binding to the TRE promoter in the indicator mice for indicator expression (GCaMP6f in this transgenic diagram). This combination of dCre and tTA facilitates adjustable indicator expression probability of layer II/III pyramidal neurons by titratable TMP administration (b) GCaMP6f expression density remained high with total TMP dose administered at 50 mg/kg body weight (i; imaged using wide-field epifluorescence microscopy; solid line marks the pia, dashed line marks cortical layer boundaries) and 0.5 mg/kg body weight (ii; using confocal microscopy, single plane) (c) GCaMP6f expression with total TMP dose administered at 0.005 mg/kg body weight (i), 0.03 mg/kg body weight (ii), and 750 mg/kg body weight (iii) TMP concentrations resulted in different levels of GCaMP6f expression density (confocal microscopy). DAPI nuclei-counterstaining shows, in addition to pyramidal neurons, dense cell population including glia, GABAergic interneurons and blood vessel-related cells. Scale bar = 50 µm; stack thickness = 15 µm.