Functional fluorescent Ca2+ indicator proteins in transgenic mice under TET control

Abstract

Genetically encoded fluorescent calcium indicator proteins (FCIPs) are promising tools to study calcium dynamics in many activity-dependent molecular and cellular processes. Great hopes-for the measurement of population activity, in particular-have therefore been placed on calcium indicators derived from the green fluorescent protein and their expression in (selected) neuronal populations. Calcium transients can rise within milliseconds, making them suitable as reporters of fast neuronal activity. We here report the production of stable transgenic mouse lines with two different functional calcium indicators, inverse pericam and camgaroo-2, under the control of the tetracycline-inducible promoter. Using a variety of in vitro and in vivo assays, we find that stimuli known to increase intracellular calcium concentration (somatically triggered action potentials (APs) and synaptic and sensory stimulation) can cause substantial and rapid changes in FCIP fluorescence of inverse pericam and camgaroo-2.

Figure 1. Genetic Designs of the FCIPs and the TET System

(A) Genetic design of fluorescence Ca²⁺ indicator proteins: (i) yellow fluorescent protein, (ii) Cg2, (iii) IP, and (iv) came-leon YC3.12. (B) Operating principles of the TET regulatory system (for details see Gossen and Bujard 2002). “L” indicates short linker sequence.

Figure 2. Expression and Functional Tests in Cell Culture

(A) HeLa cells expressing tTA and Ptetbi-luciferase/Cg2 or Ptetbi-luiferase/IP and imaged by confocal microscopy. Top row: low [Ca²⁺] (0.1 μM), bottom row: high [Ca²⁺] (25 mM) and ionomycin. Relative fluorescence changes are indicated as %ΔF/F. (B) Ratios (rlu-FL/rlu-RL) of FL to RL activity measured in mouse ear fibroblast cell cultures from all DNA-positive founders in the absence (red) and presence (green) of Dox (see Materials and Methods). Circles (solid and open) indicate the lines that were selected for crossing to the transactivator lines. Solid circles indicate lines that showed smooth fluorescence.

Figure 3. Doxycycline and tTA-Dependent FCIP Expression

Immunohistochemical assay (A–F) using rabbit polyclonal GFP antibodies/peroxidase-DAB system: (A) YC3.12, single-positive (MTH-YC3.12-7), double-positive (MTH-YC3.12-7, αCamKII-tTA), and Dox-treated double-positive (MTH-YC3.12-7, αCamKII-tTA). (B) Cg2, single-positive (MTH-Cg2-7) and doubles-positive (MTH-Cg2-7, αCamKII-tTA). (C) IP, single-positive (MTH-IP-12) and double-positive (MTH-IP-12, αCamKII-tTA). (D) Moderate-expression line of Cg2 (MTH-Cg2-14, αCamKII-tTA). (E) Low-expression line (MTH-Cg2-15, αCamKII-tTA). (F) FCIP distribution in various brain areas. (G) Fluorescence in fixed brain slices from the accessory and the main olfactory bulb. (H–K) Two-photon images of acute, living brain slices. (H) Neurons in both CA1 and striatum usually show nuclear exclusion. (I) punctate expression in low-expressing lines (also see Figure 2B, open circles); example from CA1 and cortex. Maximum intensity projection of two-photon 3D stacks taken from a brain slice (J) and a whole-mount retina (K).

Figure 4. Fluorescence Spectra and FCIP Mobility

(A) Fluorescence distribution and emission spectra of Cg2 in cultured HEK cells and in neurons (dendrites and soma) in an acute brain slice (MTH-Cg2-7). (B) Punctate fluorescence and corresponding emission spectra (MTH-Cg2-7). “*” denotes emission spectrum of a punctate fluorescence in a different brain slice (not shown). Note smooth and punctate fluorescence also in the two-photon image on the right (MTH-Cg2-14). (C) Punctate fluorescence in a double-negative littermate of MTH-Cg2-7. (D) Image (left) and emission spectrum (right) of two-photon-excited fluorescence in an acute brain slice (MTH-Cg2-7). (E) Indicator mobility by two-photon fluorescence photobleaching recovery (IP, MTH-IP-12).

Figure 5. In Vivo Two-Photon Imaging Through the Thinned Skull

Yellow cameleon 3.12 at different depths (MTH-YC3.12-8) (A) and with high resolution (MTH-YC3.12-7) (B). (C) IP at different depths (MTH-IP-1).

Figure 6. FCIP Responses to Direct and to Synaptic Stimulation in Acute Brain Slices

(A) Whole-field imaged responses of Cg2-positive cells in cortex to bursts of APs evoked by somatic current injection (whole-cell recording electrode indicated schematically); responses in the recorded (red) and in a nonrecorded (green) soma and in a region with no cell body (blue). (B) Two-photon line scan (lower trace) through the soma of a hippocampal CA1 pyramidal neuron during a burst of APs evoked by somatic current injection through a high-resistance microelectrode. (C) Whole-field-imaged responses to synaptic stimulation in cortex (five pulses at 100 Hz, 10 μA); ΔF/F image is shown below. Fluorescence and voltage responses with and without pharmacological block of glutamate channels (note suppression of APs and unmasking of inhibitory synaptic potentials).

Figure 6. Continued

(D–F) Two-photon-imaged responses to synaptic stimulation in the hippocampus. (D) CA1 region with Schaffer collateral stimulation (eight individual response traces and the averaged trace are shown, region of interest indicated in the “response” image). Averaged images (five frames) during rest and response, and their difference, respectively. In localized hot spots, responses reach 100% (panels and traces shown below). (E) Similar response amplitudes and kinetics are seen in the dentate gyrus with mossy fiber stimulation (note that the number of stimuli was only 20). (F) IP responses recorded by a two-photon line scan through a CA1 soma; stimulation (20 pulses at 200 Hz) of neurites (approximately 50 μm away from the somata).

Figure 7. Light-Evoked Ca²⁺ Responses in Retinal Ganglion Cells

(A) Intact, light-sensitive retinal whole mount with Sulforhodamine 101 (red) in the extracellular space. Blood vessels are red; IP-positive (MTH-IP-12) retinal ganglion cells are green; and unstained ganglion cells are dark. (Scale bar: 50 μm). (B) Projection of an image stack reveals the IP-labeled primary dendrites of the retinal ganglion cells. (C) Time course of Ca²⁺ response measured by high repetition rate image scan (62.5 Hz) of a soma: The cell responds with a decrease in fluorescence to the onset of the laser (asterisk) and to the repeated light stimulation (arrows). (D) Averaged (four repetitions) light-stimulus-evoked Ca²⁺ response (black trace; gray traces are single trials) measured in the soma (above) and in the primary dendrite (below) of a retinal ganglion cell.

Figure 8. In Vivo Imaging of Odor-Evoked Ca²⁺ Signals with Transgenic Indicators in the Olfactory Bulb

(A–C) IP (MTH-IP-12). (A) Raw fluorescence image. (B) Time course of fluorescence signal in the corresponding regions outlined in (C) (matching line colors). The black trace shows respiratory activity. (C) Color-coded map showing the relative change in fluorescence evoked by different odors in each pixel during the first second of the odor response. (D–F) Cg2 (MTH-Cg2-19). (D) Raw fluorescence image. (E) Time course of fluorescence signal in the corresponding regions outlined in (F) (matching line colors). (F) Color-coded maps showing the relative change in fluorescence evoked by different odors in each pixel during the first second of the odor response.