In Vivo Imaging of the Coupling between Neuronal and CREB Activity in the Mouse Brain

Abstract

Sensory experiences cause long-term modifications of neuronal circuits by modulating activity-dependent transcription programs that are vital for regulation of long-term synaptic plasticity and memory. However, it has not been possible to precisely determine the interaction between neuronal activity patterns and transcription factor activity. Here we present a technique using two-photon fluorescence lifetime imaging (2pFLIM) with new FRET biosensors to chronically image in vivo signaling of CREB, an activity-dependent transcription factor important for synaptic plasticity, at single-cell resolution. Simultaneous imaging of the red-shifted CREB sensor and GCaMP permitted exploration of how experience shapes the interplay between CREB and neuronal activity in the neocortex of awake mice. Dark rearing increased the sensitivity of CREB activity to Ca²⁺ elevations and prolonged the duration of CREB activation to more than 24 h in the visual cortex. This technique will allow researchers to unravel the transcriptional dynamics underlying experience-dependent plasticity in the brain.

Keywords: CREB; FLIM; FRET; in vivo imaging.

Figure 1:. Design and validation of a CREB FLIM biosensor

(A) Schematic design of the CREB FLIM sensor for G-CREB and R-CREB. (B-C) Representative images of pseudo-colored FLIM before and 40 min after induction of CREB activation by elevation of cAMP (25 μM forskolin and 100 μM IBMX), in HEK-293 cells transfected with G-CREB donor or S133A mutant (B) or with R-CREB and S133A mutant (C). Scale bar, 20 μm. (D) Average time course of ΔBF in HEK cells following forskolin/IBMX application for G-CREB and S133A sensors. (E) Same as (C) but for R-CREB sensor. (F) Quantification of mean ΔBF following 30 – 45 minutes from forskolin/IBMX application in HEK cells (G-CREB: 0.135 ± 0.008 and 0.0186 ± 0.005 for G-CREB and S133A respectively, n=28 for each, p < 0.0001, R-CREB: 0.09 ± 0.004 and 0.013 ± 0.005 for R-CREB and S133A respectively, n = 26 and 32 cells, ****p < 0.0001, two-tailed unpaired t-test. (G) Average time course of ΔBF of G-CREB in HEK cells following forskolin application (25 μM), followed by response reversal following application of NKY 80 (100 μM), adenylyl cyclase inhibitor. Red and blue lines represent single exponential fits to increase or decrees in binding, half-time of 11.7 and 22.8 min, respectively. (H) Top: Hippocampal CA1 cell co-expressing R-CREB (magenta) and GCaMP6s (green) merged intensity image, before and after NMDA (20μM) application. Bottom: pseudo-colored FLIM before and 5 minutes after NMDA application. Scale bar- 20μm. (I) Average time course (blue) and individual cell traces (grey) of ΔBF of R-CREB sensor following NMDA application. Black line is single exponential fit to increase in binding, half-time is 2.1 minutes. (J) Top: experimental setup for depolarization induced CREB/Ca²⁺ measurement. Bottom: CA1 cell expressing GCaMP/R-CREB before and during stimuli. Scale bar- 20 μm. (K) ΔF/F₀ for GCaMP6s response during stimulation, n=16 cells. (L) Lifetime pseudo-colored images for same cell as in (J), for R-CREB before and after stimuli. Scale bar, 20 μm. (M) Average time-course of R-CREB ΔBF following depolarization, red line is exponential fit. Half-life is 34.2 s, n = 16/16 cells/slices. **** p < 0.0001, two tailed unpaired t-test, Error bars indicate SEM for all panels.

Figure 2:. In vivo two-photon FLIM of CREB activity in L2/3 cortical cells.

(A) Representative in vivo image of G-CREB expression in L2/3 cells in the somatosensory cortex, for mEGFP-CREB (green), mCh-KIX-mCh (magenta) and merged overlay. Scale bar, 100 μm. (B) Representative traces of whole cell current clamp recordings from L2/3 pyramidal neurons in acute coronal cortical slices expressing control AAV (CyRFP) or G-CREB sensor at three different current steps (−100, 0 and 200 pA). (C) Mean number of spikes evoked by increasing depolarizing current steps. n=17/7 and 23/9 (cells/animals) for G-CREB sensor and control cells respectively, p = 0.78, two-tailed unpaired t-test. (D) Pseudo-colored FLIM images of L2/3 cells expressing either WT G-CREB or S133A mutant. Scale bar is 50 μm. (E) Comparison of in vivo BF values for G-CREB and S133A CREB sensor. Average BF is 0.18 ± 0.003 and 0.108 ± 0.0009, n = 250/4 and 212/4 (cells/animals) for G-CREB and S133A, respectively (****p < 0.0001, two-tailed unpaired t-test). (F) Correlation between mEGFP-CREB photon number and BF for the same cells in (E), Spearman correlation values are 0.11 (p = 0.09) for WT and 0.11 (p = 0.1) for S133A, respectively. (G) Representative pseudo-colored in vivo FLIM images of CREB activity in the same cells over 2 days for WT and S133A sensor, scale bar 20 μm. (H) Quantification of changes in BF over 2 imaging session, n=164/3 and 124/3 cells/animals for WT and S133A sensor, respectively. (I) Normalized ΔBF for same population of cells as in (H), average change is 0.003±0.02 and 0.001±0.001 for WT and S133A, respectively. (J) Representative images of pseudo-colored FLIM before and 40 min after acute induction of CREB activation in vivo by inclusion of 1 mM Forskolin in ACSF through a hole drilled in cranial window coverglass, scale bar, 50 μm. (K) Average time course of mean ΔBF in cortical neurons in vivo following forskolin application for G-CREB and S133A sensor. (L) Quantification of mean ΔBF following 30–45 minutes from forskolin application, 0.117 ± 0.008, n=41 for G-CREB and 0.021 ± 0.006, n=34 for WT and S133A from 3 and 2 different animals, p < 0.0001, two-tailed unpaired t-test. ****p < 0.0001, Error bars indicate SEM for all panels.

Figure 3:. CREB dynamics in the somatosensory cortex following sensory enrichment

(A) Experimental design for monitoring the effect of enriched environment on CREB activity in the somatosensory cortex. HC: home cage, EE: enriched environment. (B) Representative pseudo-colored FLIM images of the CREB sensor in the same population of cells over 3 days interval during imaging sessions in HC₁, HC₂ and EE₃. (C) Quantification of ΔBF in single cells to HC₁ for HC₁-HC₂-EE, n = 83/3 (cells/animals). One way ANOVA followed by Tukey’s multiple comparisons test. (D) Alternate experimental design for monitoring effect of enriched environment on ongoing CREB activity. (E) Representative pseudo-colored images of FLIM of CREB sensor in the same population of cells over 3 days interval during imaging sessions in HC₁, EE₂ and HC₃. (F) Quantification of ΔBF in single cells over days for HC₁-EE₂-HC₃, n = 83/3 (cells/animals). One way ANOVA followed by Tukey’s multiple comparisons test. (G) Same as E only for CREB sensor with S133A mutation. Scale bar, 50μm for all images. (H) Same as (F) only for G-CREB sensor with the S133A mutation, n = 87/4 (cells/animals). One way ANOVA followed by Tukey’s multiple comparisons test. (I) ΔBF normalized to CREB activity in HC in the first imaging session. Average change for G-CREB of 0.006±0.003, 0.053±0.04, 0.045±0.004 and 0.005±0.003 for HC₁-HC₂, HC₁-EE₃, HC₁-EE₂ and HC₁-HC₃ respectively, for S133A 0.0002±0.001 and −0.0007±0.001 for HC₁-EE₂ and HC₁-HC₃ respectively. One way ANOVA followed by Sidak’s multiple comparisons test. n.s, p>0.05, **** p < 0.0001. Error bars represent SEM.

Figure 4:. Simultaneous in vivo imaging of red-shifted CREB sensor and GCaMP6

(A) Emission spectra of mCyRFP2 and mEGFP with dotted lines indicating green and red band-pass filter ranges. (B) Example image of mCyRFP2-CREB field of view during in vivo imaging session for the 1^st, 5^th and 20^th minute of an imaging session during consecutive 7.8Hz frame imaging. Scale bar, 100 μm. (C) Normalized changes in mean fluorescence (black line) and individual cells (grey lines) across imaging sessions, n = 87/3 (cells/animals). Average fluorescence after 1, 5 and 20 min was 102.6±0.4, 104.9±1.0 and 91.37±1.2%, respectively.. p = 0.155 and p = 0.0001 for comparison of 1 and 5 minutes and 1 and 20 minutes, One way ANOVA followed by Dunnett’s multiple comparisons test. (D) R-CREB (magenta) GCaMP6s (green) intensity images during in vivo imaging session in the motor cortex. Scale bar, 50 μm. (E) Traces during a 5 min long imaging session showing activity profiles of cells marked in (D) for GCaMP6s transients alongside lifetime measured in red-channel. (F) R-CREB (magenta) GCaMP6s (green) intensity images during in vivo imaging session in the visual cortex. Scale bar, 50 μm. (G) Traces during a 50 seconds long imaging session showing visual evoked (blue vertical line) activity profiles of cells marked in (F) for GCaMP6s transients alongside lifetime measured in red-channel.. Errors represent SEM.

Figure 5:. In vivo imaging of experience dependent CREB activity following sensory stimulation.

(A) Illustration of the experimental paradigm: in vivo imaging of R-CREB/GCaMP6s activity in awake head-restrained mice during visual stimulation, either following normal rearing (naïve) or following dark rearing (DR). (B) Representative pseudo-colored FLIM images of R-CREB activity. Top: DR mouse without visual stimulation, middle: naïve mouse with visual stimulation, and bottom: DR mouse with visual stimulation. Images are show CREB activity before, 0.5 h, and 24 h after visual stimulation. Scale bar: 50 μm. (C) Time course of ΔBF in single cells BF across the 3 groups. Grey lines denote individual cells and colored thick lines denote average changes. Thick lines with light color denote average trace of cells with high BF (> 0.05) at 22 – 30 min. Statistics are for ΔBF averaged over 22 – 30 min following visual stimuli and at 24 h, compared with baseline: p = 0.98 and p = 0.11 for DR, −stimuli (n = 133/4; cells/animals); p < 0.0001 and p = 0.08 for Naïve, + stimuli (n = 181/4); p < 0.0001 and p < 0.0001 for DR, +stimuli (n = 185/4), for 0.5h and 24h, respectively. One way ANOVA followed by Dunnett’s multiple comparison test. (D) Comparison of ΔBF across groups DR, −stimuli: −0.0006 ± 0.002 and −0.005±0.003; naïve, + stimuli: 0.035±0.003 and 0.006±0.003, DR, +stimuli: 0.062±0.003 and 0.06±0.003 for 0.5h and 24hr respectively. p < 0.0001 for all comparisons except between DR no stimuli and Naive at 24 h, p = 0.072. One way ANOVA followed by Tukey’s multiple comparison test. n.s. p > 0.05, ****p < 0.0001. Error bars represent SEM.

Figure 6:. Interplay between cumulated Ca²⁺ and CREB dynamics

(A) Representative Ca²⁺ traces of a cell from a DR animal. Shown are ΔF/F₀ responses during individual trials of a natural movie (30 trials total, 7.8Hz imaging frame rate), averaged over trials ΔF/F₀ activity (bottom left). Summed Ca²⁺ (∑ΔF/F₀) and CREB ΔBF across trials are also shown for this cell (middle). The trial-by-trial increase in CREB ΔBF is plotted with the corresponding summed Ca²⁺ (∑ΔF/F₀) (right). Data points are pseudo-colored by trial number. (B) Same as in (A) for a cell from a naïve animal. (C) Relationship between CREB ΔBF and summed Ca²⁺ across cells in DR (red) and naïve (grey) mice. Data points are mean and standard error. n = 61/4 and 114/4 (cells/animals) for DR and naïve, respectively. Also shown is average ΔBF after 30 min of visual stimulation and 24 h later (right) for cells with high visually-evoked Ca²⁺ activity (ƩΔF/F₀ > 4). n = 17/4 and 57/4 (cells/animals) for DR and naïve, respectively. (D) Relationship between CREB ΔBF and summed Ca²⁺ for cells with high (solid lines, closed circles) and low (dashed lines, open circles) CREB ΔBF (>0.05 or <0.05 at ΔBF30 min, respectively). Also shown is average ΔBF after 30 min of visual stimulation and 24 h later (right) for each of these groups for cells with high visually-evoked Ca²⁺ activity (ƩΔF/F₀ > 4). n = 9 and 8 for low and high CREB in DR mice, and 45 and 12 (cells) for low and high CREB ΔBF in naïve animals, respectively). (E) Correlation between levels of CREB ΔBF at 30 min and 24 h for cells from DR (red circles, r = 0.45, p < 0.001, n = 56) and naïve (grey circles, r = 0.2930 p = 0.002, n = 87/4, cells/animals) animals.