Genetically Targeted All-Optical Electrophysiology with a Transgenic Cre-Dependent Optopatch Mouse

Abstract

Recent advances in optogenetics have enabled simultaneous optical perturbation and optical readout of membrane potential in diverse cell types. Here, we develop and characterize a Cre-dependent transgenic Optopatch2 mouse line that we call Floxopatch. The animals expressed a blue-shifted channelrhodopsin, CheRiff, and a near infrared Archaerhodopsin-derived voltage indicator, QuasAr2, via targeted knock-in at the rosa26 locus. In Optopatch-expressing animals, we tested for overall health, genetically targeted expression, and function of the optogenetic components. In offspring of Floxopatch mice crossed with a variety of Cre driver lines, we observed spontaneous and optically evoked activity in vitro in acute brain slices and in vivo in somatosensory ganglia. Cell-type-specific expression allowed classification and characterization of neuronal subtypes based on their firing patterns. The Floxopatch mouse line is a useful tool for fast and sensitive characterization of neural activity in genetically specified cell types in intact tissue.

Significance statement: Optical recordings of neural activity offer the promise of rapid and spatially resolved mapping of neural function. Calcium imaging has been widely applied in this mode, but is insensitive to the details of action potential waveforms and subthreshold events. Simultaneous optical perturbation and optical readout of single-cell electrical activity ("Optopatch") has been demonstrated in cultured neurons and in organotypic brain slices, but not in acute brain slices or in vivo Here, we describe a transgenic mouse in which expression of Optopatch constructs is controlled by the Cre-recombinase enzyme. This animal enables fast and robust optical measurements of single-cell electrical excitability in acute brain slices and in somatosensory ganglia in vivo, opening the door to rapid optical mapping of neuronal excitability.

Keywords: optogenetics; optopatch; transgenic mice; voltage imaging.

Figure 1.

Cre-dependent Optopatch2 transgenic mice show cell-type-specific expression. A, Schematic of the genomic recombination site (top), the Optopatch targeting vector (middle), and the Optopatch construct (bottom). Green arrows indicate the primer sites for distinguishing homozygous from heterozygous mice. The insert comprised CAG::loxP-Stop-loxP-Optopatch2-WPRE. CAG is a strong universal promoter (Niwa et al., 1991). The loxP-Stop-loxP cassette is a transcriptional stop motif that can be excised by the Cre recombinase. The Optopatch2 construct comprised QuasAr2-dark mOrange2–P2A-CheRiff-eGFP. QuasAr2 is a genetically encoded near infrared voltage indicator, P2A is a self-cleaving ribosome skip sequence (Hochbaum et al., 2014), CheRiff is a blue-shifted channelrhodopsin variant, and eGFP is a fluorescent expression marker for CheRiff. WPRE is the woodchuck hepatitis virus posttranscriptional regulatory element (Madisen et al., 2012; Hochbaum et al., 2014). Red arrows indicate primer sites for detection of the Optopatch sequence. Inset, Genotyping results using G1/S12 showed 1.1 kb bands in Floxopatch^+/− and Floxopatch^+/+ genomic DNA, but not in Floxopatch^−/− DNA. Genotyping results using GT5/GT8 showed 242 bp bands in Floxopatch^−/− and Floxopatch^+/− genomic DNA, but not in Floxopatch^+/+ DNA. B, Genetically targeted Optopatch expression in the CNS. Top, Sagittal sections of Floxopatch mouse brains with different Cre drivers. Confocal fluorescence images show distribution of CheRiff-eGFP. Red squares indicate the cortical area magnified in the bottom. Scale bar, 1 mm. Bottom, Confocal fluorescence images showing cellular structures. Scale bar, 50 μm. C, Genetically targeted Optopatch2 expression in DRG. Images show maximum projections of confocal z-stacks of CheRiff-eGFP fluorescence. Scale bar, 50 μm.

Figure 2.

Tests for physiological effects of transgenic Optopatch2 expression. A, Weight comparison among Floxopatch-expressing mice and controls at weaning (P21). B–E, Comparison of electrophysiological parameters in control and Optopatch2-expressing neurons from transgenic mice.

Figure 3.

Optopatch measurements in cultured DRG neurons derived from Floxopatch mice. A, Simultaneous fluorescence (red) and manual patch clamp (black) recordings during perfusion with 1 μm capsaicin of a cultured DRG neuron derived from a Na_V1.8-Cre^+/−; Floxopatch^+/− mouse (λ_exc = 640 nm, 600 W/cm², λ_em = 667–742 nm). Close-ups of the boxed regions are shown to the right of each trace. B, Left, Epifluorescence images of a cultured DRG neuron derived from a Na_V1.8-Cre^+/−; Floxopatch^+/− mouse. Scale bar, 5 μm. Middle, simultaneous fluorescence (red) and manual patch clamp (black) recordings with optogenetic stimulation. The cell was stimulated with steps of blue light (λ_exc = 488 nm, 500 ms duration, 50–200 mW/cm²) and fluorescence of QuasAr2 was recorded (λ_exc = 640 nm, 600 W/cm², λ_em = 667–742 nm). Right, Close-up of the boxed region showing correspondence of the optical and electrical signals. C, Optically evoked action potentials were recorded simultaneously via QuasAr2 fluorescence sampled at 500 Hz and manual patch-clamp electrophysiology in current-clamp mode (i = 0) sampled at 10 kHz. Signals were aligned by spike peak and averaged to calculate a mean action potential waveform. Electrical traces were down-sampled to 500 Hz. Mean action potential waveforms in DRG neurons are shown. Left, Na_V1.8-Cre^+/−; Floxoptach^+/− mice (n = 17 cells). Middle, MrgA3-Cre^+/−; Floxopatch^+/− mice (n = 5 cells). Right, SST-Cre^+/−; Floxopatch^+/− mice (n = 3 cells). Shading indicates SEM. D, Comparison of optically and electrically recorded action potential widths (full-width at half-maximum). Spike widths in SST⁺ neurons were too narrow to be well resolved in the optical measurements.

Figure 4.

Optopatch recordings with subcellular and multicellular resolution. A, Subcellular recordings of action potentials in a cultured DRG neuron derived from a CAG-CreEr^+/−;Floxopatch^+/− mouse after tamoxifen treatment. Left, Epifluorescence image of CheRiff-eGFP showing the whole cell. Scale bar, 10 μm. Middle, Close-up images of the boxed regions. Scale bar, 4 μm. Right, QuasAr2 fluorescence from the whole soma (blue) and the two colored regions indicated in the images. Stimulus: λ_exc = 488 nm, 10 ms pulses, repeated at 10 Hz, increasing from 600 mW/cm² to 3 W/cm² in increments of 60 mW/cm². QuasAr2 fluorescence excited at 600 W/cm². B, Wide-field simultaneous Optopatch recording from 11 cultured DRG neurons from a Na_V1.8-Cre^+/−;Floxopatch^+/− mouse. Left, Images of CheRiff-eGFP and QuasAr2 fluorescence. Scale bar, 20 μm. Right, Cells were stimulated with bursts of blue light, 2 ms pulses, repeated at 40 Hz, for 0.5 s, followed by 1 s rest, increasing intensity from 600 mW/cm² to 3 W/cm². QuasAr2 fluorescence excited at 600 W/cm². C, Comparison of eGFP fluorescence in cultured DRG neurons with Cre-dependent Optopatch2 expressed either via electroporation (top) or from a transgenic mouse (bottom). In both cases, neurons were derived from Na_V1.8-Cre^+/− mice and were cultured for 7 d after dissection. Scale bar, 100 μm. D, Measurements of optically induced spiking thresholds in electroporated or transgenic neurons. Stimulus protocol (bottom) and fluorescence traces (top). Excitability threshold was defined as the lowest light intensity that induced an action potential. Cells were stimulated with flashes of blue light, 5 ms on, 95 ms off, linearly increasing intensity from 600 mW/cm² to 3 W/cm² in increments of 60 mW/cm².

Figure 5.

Optopatch recordings of neuronal excitability in acute brain slice. A, Top, Epifluorescence images of the Optopatch components in CaMKII⁺ neurons in the dentate gyrus of the hippocampus. Scale bar, 10 μm. Bottom, Fluorescence of QuasAr2 in the indicated cells in response to illumination with steps of blue light of increasing intensity from 50 to 350 mW/cm². QuasAr2 fluorescence excited at 40 mW power (800 W/cm²). B, Raster plot showing the spiking patterns of 101 neurons under a nine-step stimulation protocol. C, Top, Epifluorescence images of a SST⁺ inhibitory neuron in the cortex. Scale bar, 10 μm. Bottom, Fluorescence of QuasAr2 and the optical stimulus pattern. Optical parameters are as in A. D, Raster plot showing the spiking patterns of 59 SST⁺ neurons stimulated with the same protocol as in B. E, Comparison of time-dependent spike rates of CaMKII⁺ and SST⁺ neurons under the same stimulus protocol. F, Distributions of maximum spike rates (averaged over a 500 ms stimulus) in SST⁺ and CaMKII⁺ neurons. SST⁺ neurons reached significantly higher firing rates than CaMKII neurons (p = 1.0 × 10⁻⁸, unpaired two-sided t test). G, Distributions of adaptation ratios (averaged over all stimulus strengths) in SST⁺ and CaMKII⁺ neurons. More SST⁺ neurons than CaMKII⁺ neurons were slow adapting (the populations were significantly different by the Mann–Whitney test, p = 2.5 × 10⁻⁹). Inset, Receiver operating characteristic curve for separating CaMKII⁺ neurons from SST⁺ neurons based only on average adaptation ratio (area under the curve = 0.88). H, Optopatch measurements of neuronal stability under chronic measurement conditions. Orange indicates optically evoked and optically recorded spiking recorded with a 500 ms red illumination once per minute. Purple indicates optically evoked and optically recorded spiking with continuous red laser illumination. In both cases, the red laser illumination intensity was 2500 W/cm².

Figure 6.

Electrophysiological diversity of optically probed neurons in acute brain slices. Classifications are qualitative descriptors of firing patterns, motivated by the scheme of Markram et al. (2015). A, Firing patterns of CaMKII⁺ hippocampal granule cells under step stimulation (500 ms on, 500 ms off) at blue intensities increasing from 200 to 350 mW/cm². B, Firing patterns of SST⁺ cortical interneurons stimulated as in A.

Figure 7.

Optopatch imaging of mouse nodose ganglia ex vivo and in vivo. A, Ex vivo wide-field recording in a nodose ganglion from P14 Na_V1.8-Cre^+/−;Floxopatch^+/− mouse. Left, Wide-field epifluorescence images of CheRiff-eGFP. The bottom indicates the centers of the 23 cells recorded simultaneously. Scale bar, 10 μm. Right, Optically evoked activity of 23 cells. Blue stimulus intensities increased stepwise from 600 mW/cm² to 3 W/cm² (top left). QuasAr2 fluorescence was excited with red light at 300 W/cm². B, ATP-invoked firing of a neuron in the nodose ganglion. Left, Epifluorescence image of QuasAr2 expression. The recorded cell is shown with an asterisk. Scale bar, 10 μm. Right, QuasAr2 fluorescence before (top) and after (bottom) the addition of 10 μm ATP. ATP induced rapid tonic firing that persisted for several minutes. C, Left, In vivo epifluorescence images of the Optopatch components in an intact nodose ganglion from a P58 Na_V1.8-Cre^+/−;Floxopatch^+/+ mouse. Scale bar, 10 μm. Right, Fluorescence recordings from the indicated cells under optical stimulation at blue intensity 500 mW/cm². D, Sequential Optopatch recordings from 14 nodose ganglion neurons in vivo. Left, Diagram showing locations of all the neurons recorded. Scale bar, 100 μm. Right, Fluorescence recordings of single cells under optical stimulation at blue intensities from 30 to 50 mW/cm².