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

Open ArchivePublished:December 26, 2019DOI:https://doi.org/10.1016/j.neuron.2019.11.028

      Highlights

      • New CREB biosensors (green and red) for CREB activity
      • Chronic in vivo imaging of CREB activity in L2/3 cortical cells over days
      • Simultaneous imaging of CREB and Ca2+ in awake mice
      • Interplay between CREB-Ca+2 is modulated by sensory experience

      Summary

      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

      Introduction

      Cortical circuits are responsible for integrating sensory information from the external environment. Although neuronal activity operates on millisecond timescale, sensory experience can drive plasticity over days (
      • Holtmaat A.
      • Caroni P.
      Functional and structural underpinnings of neuronal assembly formation in learning.
      ). One major signaling pathway mediates long-term plasticity via conversion of specific neuronal activity patterns to gene transcription programs (
      • West A.E.
      • Griffith E.C.
      • Greenberg M.E.
      Regulation of transcription factors by neuronal activity.
      ,
      • Yap E.-L.
      • Greenberg M.E.
      Activity-Regulated Transcription: Bridging the Gap between Neural Activity and Behavior.
      ). Activity-dependent transcription factors play key roles in decoding synaptic activity and translating it into long-term signaling (
      • Alberini C.M.
      • Kandel E.R.
      The regulation of transcription in memory consolidation.
      ,
      • Cohen S.M.
      • Li B.
      • Tsien R.W.
      • Ma H.
      Evolutionary and functional perspectives on signaling from neuronal surface to nucleus.
      ,
      • Kandel E.R.
      The molecular biology of memory: cAMP, PKA, CRE, CREB-1, CREB-2, and CPEB.
      ). To better understand the mechanisms underlying transcription-mediated circuit plasticity during learning, it is essential to simultaneously monitor neuronal activity and transcription in a large ensemble of neurons in vivo. Recent improvements in genetically encoded calcium indicators (GECIs) (
      • Chen T.-W.
      • Wardill T.J.
      • Sun Y.
      • Pulver S.R.
      • Renninger S.L.
      • Baohan A.
      • Schreiter E.R.
      • Kerr R.A.
      • Orger M.B.
      • Jayaraman V.
      • et al.
      Ultrasensitive fluorescent proteins for imaging neuronal activity.
      ) have enabled researchers to image neuronal activity with cellular and sub-cellular resolution in vivo in behaving animals (
      • Cichon J.
      • Gan W.-B.
      Branch-specific dendritic Ca(2+) spikes cause persistent synaptic plasticity.
      ,
      • Peters A.J.
      • Chen S.X.
      • Komiyama T.
      Emergence of reproducible spatiotemporal activity during motor learning.
      ). However, transcription factor activity during learning has mainly been assessed through immunostaining in fixed brain tissue (
      • Alberini C.M.
      Transcription factors in long-term memory and synaptic plasticity.
      ); thus, studying the dynamics of transcription factors in neurons of behaving animals has not been possible.
      Among activity-dependent transcription factors, cyclic AMP (cAMP) response element binding protein (CREB) has long been implicated in synaptic plasticity, learning, and memory (
      • Bourtchuladze R.
      • Frenguelli B.
      • Blendy J.
      • Cioffi D.
      • Schutz G.
      • Silva A.J.
      Deficient long-term memory in mice with a targeted mutation of the cAMP-responsive element-binding protein.
      ,
      • Han J.-H.
      • Kushner S.A.
      • Yiu A.P.
      • Cole C.J.
      • Matynia A.
      • Brown R.A.
      • Neve R.L.
      • Guzowski J.F.
      • Silva A.J.
      • Josselyn S.A.
      Neuronal competition and selection during memory formation.
      ,
      • Pittenger C.
      • Huang Y.Y.
      • Paletzki R.F.
      • Bourtchouladze R.
      • Scanlin H.
      • Vronskaya S.
      • Kandel E.R.
      Reversible inhibition of CREB/ATF transcription factors in region CA1 of the dorsal hippocampus disrupts hippocampus-dependent spatial memory.
      ,
      • Silva A.J.
      • Kogan J.H.
      • Frankland P.W.
      • Kida S.
      CREB and memory.
      ). CREB binds to multiple recognition sites across the genome (
      • Conkright M.D.
      • Guzmán E.
      • Flechner L.
      • Su A.I.
      • Hogenesch J.B.
      • Montminy M.
      Genome-wide analysis of CREB target genes reveals a core promoter requirement for cAMP responsiveness.
      ) in response to increased intracellular Ca2+ elevation in synapses (
      • Bito H.
      • Deisseroth K.
      • Tsien R.W.
      CREB phosphorylation and dephosphorylation: a Ca(2+)- and stimulus duration-dependent switch for hippocampal gene expression.
      ,
      • Wheeler D.G.
      • Groth R.D.
      • Ma H.
      • Barrett C.F.
      • Owen S.F.
      • Safa P.
      • Tsien R.W.
      Ca(V)1 and Ca(V)2 channels engage distinct modes of Ca(2+) signaling to control CREB-dependent gene expression.
      ), the cell body (
      • Hardingham G.E.
      • Arnold F.J.L.
      • Bading H.
      Nuclear calcium signaling controls CREB-mediated gene expression triggered by synaptic activity.
      ), and the nucleus (
      • Dudek S.M.
      • Fields R.D.
      Somatic action potentials are sufficient for late-phase LTP-related cell signaling.
      ) and regulates transcription of a wide array of genes (
      • Lonze B.E.
      • Ginty D.D.
      Function and regulation of CREB family transcription factors in the nervous system.
      ). Plasticity-inducing stimuli in just a few dendritic spines can lead to CREB activation, suggesting high sensitivity of the CREB signaling pathway (
      • Zhai S.
      • Ark E.D.
      • Parra-bueno P.
      • Yasuda R.
      Long-Distance Integration of Nuclear ERK signaling triggered by activation of a Few Dendritic Spines.
      ). Therefore, CREB is considered a primary molecular junction between synaptic inputs and long-term gene transcription-dependent plasticity (
      • Kandel E.R.
      The molecular biology of memory: cAMP, PKA, CRE, CREB-1, CREB-2, and CPEB.
      ). CREB is activated by phosphorylation of serine 133 (S133), a target of Ca2+-dependent signaling such as protein kinase A (PKA) and Ca2+/calmodulin-dependent kinase (CaMK) (
      • Gonzalez G.A.
      • Montminy M.R.
      Cyclic AMP stimulates somatostatin gene transcription by phosphorylation of CREB at serine 133.
      ,
      • Yamamoto K.K.
      • Gonzalez G.A.
      • Biggs 3rd, W.H.
      • Montminy M.R.
      Phosphorylation-induced binding and transcriptional efficacy of nuclear factor CREB.
      ). Immunostaining of phospho-S133 of CREB has been widely used as a marker of CREB activation in vivo (
      • Barco A.
      • Marie H.
      Genetic approaches to investigate the role of CREB in neuronal plasticity and memory.
      ). However, to probe how sensory experience shapes CREB dynamics in individual cells in vivo, it is imperative to use an approach that allows live-cell imaging.
      Fluorescence resonance energy transfer (FRET) biosensors have revolutionized the investigation of intracellular signaling dynamics in live cells (
      • Miyawaki A.
      Visualization of the spatial and temporal dynamics of intracellular signaling.
      ). Two-photon fluorescent lifetime imaging (2pFLIM) has been extensively used for imaging FRET in light-scattering brain tissue. 2pFLIM signals are relatively insensitive to fluorescence fluctuations, local fluorophore concentration, light scattering, and movement artifacts (
      • Díaz-García C.M.
      • Mongeon R.
      • Lahmann C.
      • Koveal D.
      • Zucker H.
      • Yellen G.
      Neuronal Stimulation Triggers Neuronal Glycolysis and Not Lactate Uptake.
      ,
      • Yasuda R.
      Imaging spatiotemporal dynamics of neuronal signaling using fluorescence resonance energy transfer and fluorescence lifetime imaging microscopy.
      ). Previously, FRET sensors for CREB activity have been developed based on small peptides derived from the CREB kinase inducible domain (KID) and CREB binding domain (KIX) of CREB binding protein (CBP) (
      • Friedrich M.W.
      • Aramuni G.
      • Mank M.
      • Mackinnon J.A.G.
      • Griesbeck O.
      Imaging CREB activation in living cells.
      ,
      • Kobrinsky E.
      • Schwartz E.
      • Abernethy D.R.
      • Soldatov N.M.
      Voltage-gated mobility of the Ca2+ channel cytoplasmic tails and its regulatory role.
      ,
      • Spotts J.M.
      • Dolmetsch R.E.
      • Greenberg M.E.
      Time-lapse imaging of a dynamic phosphorylation-dependent protein-protein interaction in mammalian cells.
      ). However, because these sensors are based on synthetic peptides derived from CREB, their regulation and phosphorylation can be different from full-length CREB, and this may affect signal specificity. Furthermore, these sensors were designed for intensity ratiometric imaging, which suffers from wavelength-dependent light scattering (
      • Díaz-García C.M.
      • Mongeon R.
      • Lahmann C.
      • Koveal D.
      • Zucker H.
      • Yellen G.
      Neuronal Stimulation Triggers Neuronal Glycolysis and Not Lactate Uptake.
      ,
      • Yasuda R.
      Imaging spatiotemporal dynamics of neuronal signaling using fluorescence resonance energy transfer and fluorescence lifetime imaging microscopy.
      ). Overall, the utilization and interpretation of previous sensors’ signals is complicated, especially during in vivo imaging.
      Here we developed sensitive and specific CREB biosensors that report S133-dependent activation of full-length CREB and are optimized for in vivo imaging. We demonstrate that changes in CREB activity in single neurons in layer 2/3 somatosensory cortex can be monitored over days during an enriched environment paradigm. Importantly, we demonstrate utilization of a red-shifted CREB biosensor for simultaneous in vivo two-photon imaging of CREB activity with neuronal Ca2+ dynamics by combining our red CREB sensor with GCaMP6s, a green Ca2+ sensor (
      • Chen T.-W.
      • Wardill T.J.
      • Sun Y.
      • Pulver S.R.
      • Renninger S.L.
      • Baohan A.
      • Schreiter E.R.
      • Kerr R.A.
      • Orger M.B.
      • Jayaraman V.
      • et al.
      Ultrasensitive fluorescent proteins for imaging neuronal activity.
      ). Using this approach, we examine the interplay between CREB signaling and neuronal activity in individual neurons in the primary visual cortex. We find that dark rearing, a paradigm inducing adult cortical plasticity, greatly enhances the sensory evoked magnitude and duration of CREB activation. We find that this experience-dependent modulation of CREB activity is not associated with increased neuronal calcium but, rather, a shift in the coupling between CREB and neuronal activity. Overall, monitoring of activity-dependent molecular signaling using 2pFLIM allows in vivo interrogation of neuronal signaling and provides means to dissect the molecular dynamics governing experience-dependent plasticity in brain circuits.

      Results

      Design and Validation of a CREB FLIM Biosensor

      To measure CREB activation in living cells, we developed a sensor to monitor phosphorylation at S133, the main phosphorylation site necessary for CREB signaling (
      • Parker D.
      • Ferreri K.
      • Nakajima T.
      • LaMorte V.J.
      • Evans R.
      • Koerber S.C.
      • Hoeger C.
      • Montminy M.R.
      Phosphorylation of CREB at Ser-133 induces complex formation with CREB-binding protein via a direct mechanism.
      ,
      • Yamamoto K.K.
      • Gonzalez G.A.
      • Biggs 3rd, W.H.
      • Montminy M.R.
      Phosphorylation-induced binding and transcriptional efficacy of nuclear factor CREB.
      ). The sensor measures the binding of phosphorylated CREB with KIX peptide, which specifically increases its binding affinity to CREB only when it is phosphorylated at S133 (
      • Chrivia J.C.
      • Kwok R.P.S.
      • Lamb N.
      • Hagiwara M.
      • Montminy M.R.
      • Goodman R.H.
      Phosphorylated CREB binds specifically to the nuclear protein CBP.
      ,
      • Radhakrishnan I.
      • Pérez-Alvarado G.C.
      • Parker D.
      • Dyson H.J.
      • Montminy M.R.
      • Wright P.E.
      Solution structure of the KIX domain of CBP bound to the transactivation domain of CREB: a model for activator:coactivator interactions.
      ). By tagging full-length CREB with a donor fluorophore and KIX with acceptor fluorophores, activation of CREB is reflected as an increase in FRET and mirrors endogenous CREB activity because it retains its transcriptional activity (
      • Chao J.R.
      • Ni Y.G.
      • Bolaños C.A.
      • Rahman Z.
      • DiLeone R.J.
      • Nestler E.J.
      Characterization of the mouse adenylyl cyclase type VIII gene promoter: regulation by cAMP and CREB.
      ). Although CREB activity is additionally regulated by phosphorylation of S142 and S143 (
      • Gau D.
      • Lemberger T.
      • von Gall C.
      • Kretz O.
      • Le Minh N.
      • Gass P.
      • Schmid W.
      • Schibler U.
      • Korf H.W.
      • Schütz G.
      Phosphorylation of CREB Ser142 regulates light-induced phase shifts of the circadian clock.
      ,
      • Kornhauser J.M.
      • Cowan C.W.
      • Shaywitz A.J.
      • Dolmetsch R.E.
      • Griffith E.C.
      • Hu L.S.
      • Haddad C.
      • Xia Z.
      • Greenberg M.E.
      CREB transcriptional activity in neurons is regulated by multiple, calcium-specific phosphorylation events.
      ), CREB-KIX interaction and, thus, our sensor signal predominantly depend on S133 phosphorylation (
      • Mayr B.M.
      • Canettieri G.
      • Montminy M.R.
      Distinct effects of cAMP and mitogenic signals on CREB-binding protein recruitment impart specificity to target gene activation via CREB.
      ). We developed two versions of the sensor, a green version (G-CREB), composed of mEGFP-CREB and mCherry-KIX-mCherry (mCh-KIX-mCh), and a red version (R-CREB), composed of mCyRFP2-CREB and mMaroon1-KIX-mMaroon1 (Figure 1A). mCyRFP2 is a new fluorescent protein that has improved photostability compared with mCyRFP1 and similar long Stokes shift as well as mono-exponential fluorescent lifetime decay (Figure S1). mMaroon1 is a far-red florescent protein (
      • Bajar B.T.
      • Lam A.J.
      • Badiee R.K.
      • Oh Y.-H.
      • Chu J.
      • Zhou X.X.
      • Kim N.
      • Kim B.B.
      • Chung M.
      • Yablonovitch A.L.
      • et al.
      Fluorescent indicators for simultaneous reporting of all four cell cycle phases.
      ) that can be used as a highly efficient FRET acceptor for mCyRFP2 (Figure S1).
      Figure thumbnail gr1
      Figure 1Design and Validation of a CREB FLIM Biosensor
      (A) Schematic design of the CREB FLIM sensor for G-CREB and R-CREB.
      (B and 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 HEK293 cells transfected with the G-CREB donor or S133A mutant (B) or with R-CREB and the S133A mutant (C). Scale bar, 20 μm.
      (D) Average time course of changes in BF (ΔBF) in HEK cells following forskolin and IBMX application for G-CREB and S133A sensors.
      (E) The same as (D) but for the R-CREB sensor.
      (F) Quantification of mean ΔBF following 30–45 min 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), an adenylyl cyclase inhibitor. Red and blue lines represent single exponential fits to increases or decreases in binding; half-time of 11.7 and 22.8 min, respectively.
      (H) Top: merged intensity image of hippocampal CA1 cell co-expressing R-CREB (magenta) and GCaMP6s (green) before and after NMDA (20 μM) application. Bottom: pseudo-colored FLIM before and 5 min after NMDA application. Scale bar, 20 μm.
      (I) Average time course (blue) of ΔBF of the R-CREB sensor following NMDA application. The black line is a single exponential fit to increase in binding; half-time is 2.1 min.
      (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 the 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; the red line is the 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.
      To measure FRET changes, we used 2pFLIM (
      • Yasuda R.
      • Harvey C.D.
      • Zhong H.
      • Sobczyk A.
      • van Aelst L.
      • Svoboda K.
      Supersensitive Ras activation in dendrites and spines revealed by two-photon fluorescence lifetime imaging.
      ), a quantitative approach providing robust signals for longitudinal in vivo imaging (
      • Díaz-García C.M.
      • Mongeon R.
      • Lahmann C.
      • Koveal D.
      • Zucker H.
      • Yellen G.
      Neuronal Stimulation Triggers Neuronal Glycolysis and Not Lactate Uptake.
      ,
      • Ma L.
      • Jongbloets B.C.
      • Xiong W.-H.
      • Melander J.B.
      • Qin M.
      • Lameyer T.J.
      • Harrison M.F.
      • Zemelman B.V.
      • Mao T.
      • Zhong H.
      A Highly Sensitive A-Kinase Activity Reporter for Imaging Neuromodulatory Events in Awake Mice.
      ). Following expression of sensors in cell lines, we measured the binding fraction (BF) of G-CREB or R-CREB following stimulation of cells with application of a cocktail of forskolin and 3-isobutyl methylxanthine (IBMX), which increases intracellular cAMP levels and activates CREB. This resulted in a robust and sustained increase in the BF of the CREB sensor (Figures 1B and 1C). To validate the specificity of this FRET increase, we mutated S133 to alanine (S133A), preventing its phosphorylation. This abolished the increase in BF following the same stimulation (the changes in BF were 0.135 ± 0.008 and 0.0186 ± 0.005 for G-CREB and S133A and 0.09 ± 0.004 and 0.013 ± 0.005 for R-CREB, respectively; Figures 1B–1F). Furthermore, we could reverse the increase in sensor activity following forskolin application using an adenylyl cyclase inhibitor (NKY 80; Figure 1G; Figure S2A; half-times for activation and reversal of 11.7 and 22.8 min, respectively), suggesting specificity and reversibility of the sensor to CREB activity. To further examine CREB sensor activity in neurons, we transfected hippocampal organotypic neurons with G-CREB (Figures S2B–S2E). Application of forskolin increased the G-CREB BF in neurons, and this increase depended on S133 phosphorylation because introduction of the S133A mutation in CREB abolished the forskolin-induced BF increase (Figures S2C–S2E). To measure CREB activity following Ca2+ elevation, we applied NMDA for 2 min at near-physiological temperature (35°C) to hippocampal neurons co-expressing R-CREB and GCaMP6s (Figures 1H and 1I). We observed rapid CREB activation that plateaued within 5 min following Ca2+ influx, as monitored by GCaMP6s (Figures 1H and 1I; half-time of 2.1 min). To measure the kinetics of our CREB sensor in slices, we electrically stimulated the Schaffer collateral axons near a CA1 pyramidal cell that expressed both GCaMP and R-CREB (Figure 1J). We simultaneously monitored Ca2+ elevations and R-CREB responses during and following delivery of 350 pulses at 10 Hz (Figure 1K). The BF of R-CREB showed a rapid increase following stimuli, with a half-life of 34 s (Figures 1K and 1L) and decayed with a half-life of 9.6 min (Figure S2F). These measurements are in general agreement with rapid (seconds) depolarization-induced CREB phosphorylation at S133 (
      • Cohen S.M.
      • Ma H.
      • Kuchibhotla K.V.
      • Watson B.O.
      • Buzsáki G.
      • Froemke R.C.
      • Tsien R.W.
      Excitation-Transcription Coupling in Parvalbumin-Positive Interneurons Employs a Novel CaM Kinase-Dependent Pathway Distinct from Excitatory Neurons.
      ,
      • Cohen S.M.
      • Suutari B.
      • He X.
      • Wang Y.
      • Sanchez S.
      • Tirko N.N.
      • Mandelberg N.J.
      • Mullins C.
      • Zhou G.
      • Wang S.
      • et al.
      Calmodulin shuttling mediates cytonuclear signaling to trigger experience-dependent transcription and memory.
      ) and slower (minutes) dephosphorylation (
      • Bito H.
      • Deisseroth K.
      • Tsien R.W.
      CREB phosphorylation and dephosphorylation: a Ca(2+)- and stimulus duration-dependent switch for hippocampal gene expression.
      ), as measured by immunohistochemistry in dissociated neurons. The on-kinetics were similar to the duration of the stimulation (∼5 s), consistent with the fast on-kinetics of CREB activation previously reported (
      • Cohen S.M.
      • Ma H.
      • Kuchibhotla K.V.
      • Watson B.O.
      • Buzsáki G.
      • Froemke R.C.
      • Tsien R.W.
      Excitation-Transcription Coupling in Parvalbumin-Positive Interneurons Employs a Novel CaM Kinase-Dependent Pathway Distinct from Excitatory Neurons.
      ). Together, our results demonstrate that our CREB sensors are well suited to provide specific detection of S133-dependent CREB activity in living cells.

      In Vivo 2pFLIM

      To image CREB activity in a population of neurons, we generated adeno-associated viruses (AAVs) to express G-CREB under the neuronal synapsin-1 (SYN) promoter. Three weeks after viral injection, we observed nucleus-localized mEGFP-CREB-positive cells co-localized with mCh-KIX-mCh cells (Figure 2A; Figures S2F and S2G). Previous studies have revealed that CREB overexpression using herpes simplex virus (HSV) can cause an increase in neuronal excitability (
      • Kim J.
      • Kwon J.-T.
      • Kim H.-S.
      • Josselyn S.A.
      • Han J.-H.
      Memory recall and modifications by activating neurons with elevated CREB.
      ,
      • Zhou Y.
      • Won J.
      • Karlsson M.G.
      • Zhou M.
      • Rogerson T.
      • Balaji J.
      • Neve R.
      • Poirazi P.
      • Silva A.J.
      CREB regulates excitability and the allocation of memory to subsets of neurons in the amygdala.
      ). We therefore performed whole-cell patch-clamp recordings of cortical neurons infected with AAV encoding G-CREB or CyRFP1 (control, Ctrl) to examine electrophysiological properties. We did not find changes in synaptic transmission, firing rates, sag, after-hyperpolarization (AHP) and spike properties of neurons expressing G-CREB compared with control neurons in acute cortical slices (Figures 2B and 2C; Figures S3A–S3D). One possible reason for this discrepancy could be the relatively lower expression levels induced by AAV compared with HSV. We quantified the levels of AAV-driven G-CREB or R-CREB using immunohistochemistry against total CREB levels, which revealed moderate levels of overexpression (∼2-fold; Figures S3E and S3F), in contrast to HSV-mediated expression, which resulted in ∼10-fold overexpression of CREB per cell (
      • Kim J.
      • Kwon J.-T.
      • Kim H.-S.
      • Josselyn S.A.
      • Han J.-H.
      Memory recall and modifications by activating neurons with elevated CREB.
      ).
      Figure thumbnail gr2
      Figure 2In Vivo 2pFLIM 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) and mCh-KIX-mCh (magenta) and a 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, Ctrl) or the 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 the 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 wild-type (WT) G-CREB or the S133A mutant. Scale bar, 50 μm.
      (E) Comparison of in vivo BF values for the G-CREB and S133A CREB sensors. 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 as in (E). Spearman correlation values are 0.11 (p = 0.09) for the 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 the WT and the S133A sensor. Scale bar, 20 μm.
      (H) Quantification of changes in BF over 2 imaging sessions; n = 164/3 and 124/3 cells/animals for the WT and the S133A sensor, respectively.
      (I) Normalized ΔBF for the same population of cells as in (H); average change is 0.003 ± 0.02 and 0.001 ± 0.001 for the 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 artificial cerebrospinal fluid (ACSF) through a hole drilled in the cranial window coverglass. Scale bar, 50 μm.
      (K) Average time course of mean ΔBF in cortical neurons in vivo following forskolin application for the G-CREB and S133A sensor.
      (L) Quantification of mean ΔBF following 30–45 min 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.
      We used 2pFLIM to measure the fluorescence lifetime of individual layer 2/3 (L2/3) cell nuclei under light anesthesia (STAR Methods). 2pFLIM provides robust signals independent of target depth and intensity (Figure S4). To evaluate the capability of our approach to quantitatively measure CREB activity in single cells over days, we performed a detailed analysis of signal-to-noise characteristics of our sensor in vivo by comparing sensor lifetime and photon collection duration (∼1–60 s; Figure S5A). We found that frame-to-frame variation of the S133A mutant sensor, which should have minimal biological variation, decreased with increased measurement duration, following the theoretical curve of shot noise (Figure S5B). When we averaged the signal over 40–80 s (290–580 frames at 7.8-Hz imaging), similar to the time constant of the sensor (Figure 1), the noise levels were less than 1% BF. Although the wild-type G-CREB sensor showed a similar frame-to-frame variation as S133A, the average BF as well as cell-to-cell variations were much larger (Figures 2D and 2E; Figure S5C). The number of photons averaged did not correlate with the BF of G-CREB and S133A (Figure 2F), suggesting that sensor readout is relatively independent of expression level. In vivo 2pFLIM allowed us to repeatedly measure the fluorescent lifetime of G-CREB in the same cells over days (Figure 2G). We measured the BF from the same identified cells in two consecutive imaging sessions over the course of 24 h (Figure 2G). On average, the BF did not significantly change over days for both S133A and WT sensors, but the G-CREB sensor showed a higher degree of day-to-day cellular variability than the S133A sensor (Figures 2H and 2I; Figures S5B and S5C). To directly drive CREB activity in vivo, we topically applied forskolin onto a hole in the imaging window, which resulted in a rapid increase in BF that depended on intact S133 (BF change of 0.117 ± 0.008 and 0.021 ± 0.006 for G-CREB and S133A CREB, respectively; Figures 2J–2L). Collectively, our results demonstrate that G-CREB can report S133-dependent CREB activity in vivo.

      CREB Dynamics in the Somatosensory Cortex following Sensory Enrichment

      It has been known that environmental enrichment drives cortical plasticity in somatosensory areas (
      • Bengoetxea H.
      • Ortuzar N.
      • Bulnes S.
      • Rico-Barrio I.
      • Lafuente J.V.
      • Argandoña E.G.
      Enriched and deprived sensory experience induces structural changes and rewires connectivity during the postnatal development of the brain.
      ). Numerous signaling molecules, including CREB, have been shown to be modulated under this condition (
      • van Praag H.
      • Kempermann G.
      • Gage F.H.
      Neural consequences of environmental enrichment.
      ,
      • Rampon C.
      • Jiang C.H.
      • Dong H.
      • Tang Y.P.
      • Lockhart D.J.
      • Schultz P.G.
      • Tsien J.Z.
      • Hu Y.
      Effects of environmental enrichment on gene expression in the brain.
      ). Previous measurements of intracellular signaling during environmental enrichment have mainly used immunostaining in fixed brain tissue; thus, it has not been possible to investigate the temporal association between signaling dynamics in single cells and sensory enrichment. Therefore, we monitored CREB activity in the same population of somatosensory L2/3 cells using 2pFLIM over multiple days while mice experienced an enriched environment (Figure 3A; STAR Methods).
      Figure thumbnail gr3
      Figure 3CREB Dynamics in the Somatosensory Cortex following Sensory Enrichment
      (A) Experimental design for monitoring the effect of an enriched enviroment (EE) on CREB activity in the somatosensory cortex. HC, home cage.
      (B) Representative pseudo-colored FLIM images of the CREB sensor in the same population of cells over a 3-day 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 the effect of an EE on ongoing CREB activity.
      (E) Representative pseudo-colored images of FLIM of the CREB sensor in the same population of cells over a 3-day 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) The same as (E) but for the CREB sensor with the S133A mutation. Scale bar, 50 μm.
      (H) The same as (F) but for the 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 the 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; average change for S133A of 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., not significant (p > 0.05); ∗∗∗∗p < 0.0001. Error bars represent SEM.
      Mice kept in their home cage (HC) for a period of 3 days with minimal sensory stimuli showed little change in overall CREB activity during this period (HC1-HC2; Figures 3A–3C). We then exposed the same mice to an enriched sensory environment that included housing in larger cages containing various objects. Object type and location were changed daily for a period of 3 days before a third imaging session (enriched environment [EE]3; Figures 3A–3C). Subsequent imaging of the same population of cells revealed a significant increase in overall CREB activity following EE exposure (changes in average BF across cells of 0.053 ± 0.004 and 0.006 ± 0.003 for HC1-EE3 and HC1-HC2, respectively; Figures 3B, 3C, and 3I). To rule out non-specific effects, we performed chronic imaging of G-CREB in mice that were transferred from the HC after the first imaging session to the EE before a second imaging session. Again, we could reliably detect an increase in overall CREB activity following EE stimuli (change of 0.045 ± 0.004 for HC1-EE2; Figures 3D–3F and 3I). Interestingly, subsequent transfer of mice back to the HC environment for 3 days resulted in a reduction in CREB activity levels to the baseline level (change of 0.005 ± 0.003 between HC1-HC3; Figures 3D–3F and 3I). In contrast, in vivo imaging of the S133A CREB sensor did not show appreciable changes in BF following EE exposure (change of 0.0002 ± 0.001 and −0.0007 ± 0.001 for HC1-EE2 and HC1-HC3, respectively; Figures 3G–3I). These results demonstrate that in vivo 2pFLIM imaging of CREB sensors allows single cell monitoring of CREB activity in response to alteration of sensory experience.

      Simultaneous CREB and GCaMP Two-Photon Imaging

      To fully understand the coupling between neuronal activity and transcription, it is crucial to simultaneously record ongoing CREB activity together with neuronal activity. The R-CREB sensor can be simultaneously and separately imaged alongside the highly sensitive GFP-based GECI GCaMP6 (
      • Chen T.-W.
      • Wardill T.J.
      • Sun Y.
      • Pulver S.R.
      • Renninger S.L.
      • Baohan A.
      • Schreiter E.R.
      • Kerr R.A.
      • Orger M.B.
      • Jayaraman V.
      • et al.
      Ultrasensitive fluorescent proteins for imaging neuronal activity.
      ). This combination permitted spectral separation of GCaMP6s and R-CREB under 920-nm two-photon excitation in hippocampal slices (Figure 4A; Figures S6A–S6C). We then tested simultaneous imaging of R-CREB and GCaMP in vivo. Cells in the L2/3 motor cortex were infected with AAV encoding these sensors for in vivo 2pFLIM while mice were head-fixed on a custom-built running disk with adjustable counterweights (
      • Prevedel R.
      • Verhoef A.J.
      • Pernía-Andrade A.J.
      • Weisenburger S.
      • Huang B.S.
      • Nöbauer T.
      • Fernández A.
      • Delcour J.E.
      • Golshani P.
      • Baltuska A.
      • Vaziri A.
      Fast volumetric calcium imaging across multiple cortical layers using sculpted light.
      ; Figures 4B–4E). Following a habituation period, mice could run on the disc during minutes-long imaging sessions. The improved properties of mCyRFP2 allowed continuous imaging of the R-CREB sensor alongside GCaMP6s in vivo at an 8-Hz frame rate for more than 20 min with minimal photobleaching (single-cell fluorescence of 102.6% ± 0.4%, 104.9% ± 1.0%, and 91.37% ± 1.2% after 2, 5, and 20 min of imaging, respectively; Figures 4B and 4C). GCaMP6s calcium transients and R-CREB FLIM signals within individual cells exhibited no detectable cross-talk during imaging sessions (Figures 4D and 4E). Importantly, simultaneous imaging enabled us to directly compare in vivo Ca2+ dynamics of L2/3 cells expressing both R-CREB and GCaMP6s with neighboring cells expressing only GCaMP6s. We observed no difference in Ca2+ signals between cells with and without R-CREB expression in awake mice (Figure S6D), corroborating our electrophysiological measurements (Figures 2B and 2C; S3A–S3D). This combination also enabled us to image CREB-Ca2+ interplay in the L2/3 visual cortex of awake mice during visual stimulation. Again, spectral separation permitted detection of a FLIM signal in the red emission channel alongside visually evoked calcium transients with no detectable cross-talk (Figures 4F and 4G). Altogether, this approach enables precise measurements of the coupling between neuronal activity and transcription factor activity in vivo in awake animals.
      Figure thumbnail gr4
      Figure 4Simultaneous In Vivo Imaging of the R-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 an mCyRFP2-CREB field of view during in vivo imaging session for the first, fifth, and 20th min of consecutive 7.8-Hz-frame imaging. Scale bar, 100 μm.
      (C) Normalized changes in mean fluorescence (black line) and individual cells (gray 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 min and 1 and 20 min; 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 imaging session show activity profiles of the cells marked in (D) for GCaMP6s transients alongside lifetime measured in the 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-s imaging session show visually evoked (blue vertical lines) activity profiles of the cells marked in (F) for GCaMP6s transients alongside lifetime measured in the red channel.

      Experience-Dependent CREB Dynamics in the Primary Visual Cortex

      Following validation of simultaneous imaging of CREB and Ca2+ activity in vivo, we set out to quantify how these intracellular signals are coupled in the L2/3 visual cortex and how this coupling depends on sensory experience. In the visual cortex, dark rearing (DR) of adult animals induces changes in activity-dependent signaling that leads to the regulation of gene transcription (
      • Tropea D.
      • Kreiman G.
      • Lyckman A.
      • Mukherjee S.
      • Yu H.
      • Horng S.
      • Sur M.
      Gene expression changes and molecular pathways mediating activity-dependent plasticity in visual cortex.
      ). Previous studies have found that DR induces a shift in plasticity state through a reduced and increased threshold for N-methyl-d-aspartate (NMDA)-dependent long-term potentiation (LTP) and long-term derpression (LTD), respectively (
      • Philpot B.D.
      • Espinosa J.S.
      • Bear M.F.
      Evidence for altered NMDA receptor function as a basis for metaplasticity in visual cortex.
      ), and correlated with increased ocular dominance plasticity (
      • He H.-Y.H.-Y.
      • Hodos W.
      • Quinlan E.M.
      Visual deprivation reactivates rapid ocular dominance plasticity in adult visual cortex.
      ). Thus, we postulated that the relationship between Ca2+ activity and CREB signaling in the visual cortex could be shaped by DR. We placed adult (older than postnatal day 45 [P45]) mice in the dark for 7 days and examined Ca2+ and CREB activity in vivo in L2/3 visual cortical neurons in awake mice before, during, and after repeated presentations of a natural video sequence (Figure 5A). Visual stimulation was performed in the dark, so sensory experience was precisely controlled. We found that, on average, L2/3 cortical neurons of DR mice displayed a robust increase in CREB activity over the 30 min of visual stimulation (Figures 5B–5D, “DR, +stimuli”). This elevated CREB activity persisted for at least 24h (Figure 5B-D). In contrast, control DR mice, who did not receive visual stimulation, displayed no average change in CREB activity (Figure 5B-D, DR, −stimuli). In naive mice (housed in normal dark-light settings), CREB activity increased in response to visual stimulation, but at a significantly lower level compared with DR mice, and CREB activity returned to the baseline after 24 h (Figures 5B–5D, Naive, +stimuli, orange). In naive animals, a small population of cells did show high activation of CREB at 30 min (BF, >0.05) (Figure 5C, center, light orange). However, these cells showed reversal of the BF at 24 h, suggesting that the persistence of CREB activation is driven by a shift in plasticity state following DR. In addition, repeated measurements of the R-CREB BF before and 7 days after DR revealed no change in basal BF (Figure S6E), and there was no significant difference in BF between DR and naive mice prior to visual stimulation (Figure S6F). These results suggest that both the amplitude and persistence of cellular CREB activity in response to visual stimulation are regulated by sensory experience.
      Figure thumbnail gr5
      Figure 5In 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 (naive) or following dark rearing (DR).
      (B) Representative pseudo-colored FLIM images of R-CREB activity. Top: a DR mouse without visual stimulation. Center: a naive mouse with visual stimulation. Bottom: a DR mouse with visual stimulation. Images show CREB activity before, 0.5 h after, and 24 h after visual stimulation. Scale bar, 50 μm.
      (C) Time course of ΔBF of single cells across the 3 groups. Grey lines denote individual cells, and colored thick lines denote average changes. Thick lines with a light color denote average traces of cells with a 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 naive, + stimuli (n = 181/4); p < 0.0001 and p < 0.0001 for DR, +stimuli (n = 185/4) for 0.5 h and 24 h, 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; naive, +stimuli: 0.035 ± 0.003 and 0.006 ± 0.003; DR, +stimuli: 0.062 ± 0.003 and 0.06 ± 0.003 for 0.5 h and 24 h, respectively. p < 0.0001 for all comparisons except between DR −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.
      Because CREB is known to be activated by Ca2+ elevation (
      • Sheng M.
      • Thompson M.A.
      • Greenberg M.E.
      CREB: a Ca(2+)-regulated transcription factor phosphorylated by calmodulin-dependent kinases.
      ), the observed changes by DR could be caused by differences in visual activity in individual cells. To address this, we examined Ca2+ and CREB activity during visual stimulation in cells co-expressing GCaMP6s and R-CREB. As demonstrated in two example cells (Figures 6A and 6B ), individual presentations of the natural video sequence evoked selective Ca2+ responses. Because CREB is likely an integrator of somatic Ca2+ influx (
      • Hardingham G.E.
      • Arnold F.J.L.
      • Bading H.
      Nuclear calcium signaling controls CREB-mediated gene expression triggered by synaptic activity.
      ), we compared the cumulative sum of Ca2+ with changes in CREB activity (Figure 6C). Despite slightly higher accumulation of Ca2+ activity in naive animals (integrated Ca2+: median ƩΔF/F = 3.63 and 4.16, respectively; p < 0.001, Mann-Whitney test), cells in DR animals displayed larger increases in CREB BF than cells in naive animals in response to similar levels of integrated Ca2+ (Figures 6C, S6G, and S6H). In addition, we did not find any difference in cumulative Ca2+ during visual stimulation in cells co-expressing GCaMP and R-CREB compared with cells expressing GCaMP alone in the same fields of view (Figure S6I). We then grouped cells based on their CREB BF levels at 30 min of visual stimulation into high- and low-activity groups. Cell populations with high changes in CREB BF (>0.05) exhibited a similar trend in the relationship between CREB activity and integrated Ca2+ during the first 30 min of stimuli for both DR and naive animals (Figure 6D). However, CREB activity in DR mice, but not in naive mice, maintained that level of activity 24 h later, irrespective of the degree of activation during visual stimulation (Figure 6D, right). Consistent with the sustained activity of CREB in DR mice, the increase in CREB BF following 30 min of visual stimuli was strongly correlated with CREB BF levels 24 h later in DR mice (Spearman’s r = 0.45, p < 0.001), although we also observed a smaller significant correlation in in the naive group (Spearman’s r = 0.30, p = 0.002) (Figure 6E). These data suggest that the coupling between neuronal and CREB activity is modified by sensory experience and provide a potential mechanism for experience-dependent shifts in the plasticity state of visual cortical circuits.
      Figure thumbnail gr6
      Figure 6Interplay between Accumulated 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 video (30 trials total, 7.8-Hz imaging frame rate), averaged over trial ΔF/F0 activity (bottom left). Summed Ca2+ (∑ΔF/F0) and CREB ΔBF across trials are also shown for this cell (center). 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) The same as in (A) for a cell from a naive animal.
      (C) Relationship between CREB ΔBF and summed Ca2+ across cells in DR (red) and naive (gray) mice. Data points indicate SEM. n = 61/4 and 114/4 cells/animals for DR and naive, respectively. Also shown is the 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 naive, 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 ΔBF 30 min, respectively). Also shown is the 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 naive 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 naive (gray circles, r = 0.2930 p = 0.002, n = 87/4 cells/animals) animals.

      Discussion

      Here we report the development and implementation of in vivo 2pFLIM for chronic monitoring of neuronal transcription factor activity in the mouse cortex. Using a novel CREB sensor, we demonstrated that individual cells in the somatosensory cortex undergo dynamic changes in CREB activity over days, positively associated with sensory enrichment. We engineered a red-shifted CREB sensor that can be combined with green GECIs, to provide simultaneous optical readouts of transcription factor activity and calcium activity in awake animals. This enabled us to establish experience-dependent coupling between CREB signaling and neuronal activity in vivo. This approach could be generalized to utilize various biosensors for a wide range of signaling molecules important for synaptic plasticity.

      New CREB Sensor Measures S133-Dependent CREB Activity

      Several sensors for CREB have been developed previously. All of them are based on the interaction of KIX and KID; thus, these sensors likely report the activity of kinases that can phosphorylate KID peptide in the nucleus (like PKA and CaMKIV;
      • Shaywitz A.J.
      • Greenberg M.E.
      CREB: a stimulus-induced transcription factor activated by a diverse array of extracellular signals.
      ). In contrast, our sensors directly monitor the binding of full-length CREB with KIX via S133 phosphorylation; thus, these sensors would be regulated similarly as endogenous CREB. In this study, we characterized detection of rapid CREB activity using our sensor following electrical stimulation in hippocampal organotypic slices (half-life of 34 s, similliar to the duration of the simulation, 35 s). Previous immunofluorescence measurements in dissociated neuronal cultures have indicated a faster value for endogenous CREB S133 phosphorylation (∼8 s;
      • Cohen S.M.
      • Ma H.
      • Kuchibhotla K.V.
      • Watson B.O.
      • Buzsáki G.
      • Froemke R.C.
      • Tsien R.W.
      Excitation-Transcription Coupling in Parvalbumin-Positive Interneurons Employs a Novel CaM Kinase-Dependent Pathway Distinct from Excitatory Neurons.
      ).Therefore, utilization of the CREB sensor for precise kinetic analysis may require further validation. Another potential limitation of our CREB sensor is that, because it uses an active transcription factor, it may interfere with endogenous signaling pathways. Indeed, previous studies have found that strong overexpression of CREB resulted in increased neuronal excitability (
      • Kim J.
      • Kwon J.-T.
      • Kim H.-S.
      • Josselyn S.A.
      • Han J.-H.
      Memory recall and modifications by activating neurons with elevated CREB.
      ,
      • Zhou Y.
      • Won J.
      • Karlsson M.G.
      • Zhou M.
      • Rogerson T.
      • Balaji J.
      • Neve R.
      • Poirazi P.
      • Silva A.J.
      CREB regulates excitability and the allocation of memory to subsets of neurons in the amygdala.
      ) and could further shift the behavioral performance of animals in learning paradigms (
      • Han J.-H.
      • Kushner S.A.
      • Yiu A.P.
      • Cole C.J.
      • Matynia A.
      • Brown R.A.
      • Neve R.L.
      • Guzowski J.F.
      • Silva A.J.
      • Josselyn S.A.
      Neuronal competition and selection during memory formation.
      ,
      • Kida S.
      • Josselyn S.A.
      • Peña de Ortiz S.
      • Kogan J.H.
      • Chevere I.
      • Masushige S.
      • Silva A.J.
      CREB required for the stability of new and reactivated fear memories.
      ,
      • Lisman J.
      • Cooper K.
      • Sehgal M.
      • Silva A.J.
      Memory formation depends on both synapse-specific modifications of synaptic strength and cell-specific increases in excitability.
      ). In this study, we used a relatively mild promoter (SYN) and AAVs producing an ∼2-fold increased expression level (Figures S3E and S3F). Under these conditions, we did not observe any detectable effects of CREB overexpression in slices by comparing cellular electrophysiological properties (Figures 2 and S3A–S3D) and in vivo by measuring spontaneous and evoked calcium transients in cells expressing the CREB senor (Figures S6D and S6I). This is also consistent with a study showing that CREB overexpression with AAV does not alter excitability in the adult mouse hippocampus (
      • Yu X.-W.
      • Curlik D.M.
      • Oh M.M.
      • Yin J.C.
      • Disterhoft J.F.
      CREB overexpression in dorsal CA1 ameliorates long-term memory deficits in aged rats.
      ). Future utilization of recent CRISPR-Cas9-dependent knockin strategies (
      • Mikuni T.
      • Nishiyama J.
      • Sun Y.
      • Kamasawa N.
      • Yasuda R.
      High-Throughput, High-Resolution Mapping of Protein Localization in Mammalian Brain by In Vivo Genome Editing.
      ,
      • Nishiyama J.
      • Mikuni T.
      • Yasuda R.
      Virus-Mediated Genome Editing via Homology-Directed Repair in Mitotic and Postmitotic Cells in Mammalian Brain.
      ) could replace overexpression of biosensors with targeted knockin of sensors based on endogenous signaling proteins.

      CREB Dynamics during Experience-Dependent Plasticity

      Ongoing changes in the pattern and rate of neuronal activity are manifested in a wide range of activity-dependent gene expression profiles (
      • Flavell S.W.
      • Greenberg M.E.
      Signaling mechanisms linking neuronal activity to gene expression and plasticity of the nervous system.
      ). In particular, previous studies have examined how changes in neuronal activity in vivo and in vitro can lead to CREB-mediated gene transcription using immunohistochemical analysis of S133 phospho-CREB (
      • Alberini C.M.
      Transcription factors in long-term memory and synaptic plasticity.
      ). In dissociated neuronal cultures, sustained synaptic activity can induce CaMKII-dependent CREB phosphorylation (
      • Bito H.
      • Deisseroth K.
      • Tsien R.W.
      CREB phosphorylation and dephosphorylation: a Ca(2+)- and stimulus duration-dependent switch for hippocampal gene expression.
      ,
      • Ma H.
      • Groth R.D.
      • Cohen S.M.
      • Emery J.F.
      • Li B.
      • Hoedt E.
      • Zhang G.
      • Neubert T.A.
      • Tsien R.W.
      γCaMKII shuttles Ca2+/CaM to the nucleus to trigger CREB phosphorylation and gene expression.
      ). In hippocampal slices, CREB in pyramidal neurons can be activated by a burst of somatic action potentials without synaptic input (
      • Dudek S.M.
      • Fields R.D.
      Somatic action potentials are sufficient for late-phase LTP-related cell signaling.
      ) or LTP-inducing stimuli in a few dendritic spines (
      • Zhai S.
      • Ark E.D.
      • Parra-bueno P.
      • Yasuda R.
      Long-Distance Integration of Nuclear ERK signaling triggered by activation of a Few Dendritic Spines.
      ). In addition, CREB activity can be detected in various brain regions following learning paradigms or sensory stimuli (
      • Ginty D.D.
      • Kornhauser J.M.
      • Thompson M.A.
      • Bading H.
      • Mayo K.E.
      • Takahashi J.S.
      • Greenberg M.E.
      Regulation of CREB phosphorylation in the suprachiasmatic nucleus by light and a circadian clock.
      ,
      • Han J.-H.
      • Kushner S.A.
      • Yiu A.P.
      • Cole C.J.
      • Matynia A.
      • Brown R.A.
      • Neve R.L.
      • Guzowski J.F.
      • Silva A.J.
      • Josselyn S.A.
      Neuronal competition and selection during memory formation.
      ,
      • Liu F.C.
      • Graybiel A.M.
      Spatiotemporal dynamics of CREB phosphorylation: transient versus sustained phosphorylation in the developing striatum.
      ,
      • Moore A.N.
      • Waxham M.N.
      • Dash P.K.
      Neuronal activity increases the phosphorylation of the transcription factor cAMP response element-binding protein (CREB) in rat hippocampus and cortex.
      ,
      • Tropea D.
      • Kreiman G.
      • Lyckman A.
      • Mukherjee S.
      • Yu H.
      • Horng S.
      • Sur M.
      Gene expression changes and molecular pathways mediating activity-dependent plasticity in visual cortex.
      ). However, immunohistochemistry allows analysis at one time point, making it difficult to determine the temporal dynamics of CREB during specific behavior as well as variability across cells or within animals.
      The benefit of our in vivo imaging technique is highlighted by our demonstration of CREB dynamics in the somatosensory cortex during exposure to an enriched sensory environment over days (Figure 3) and simultaneous imaging of R-CREB and GCaMP in awake animals following and during sensory manipulation in the visual cortex (Figures 4 and 5). Our technique enables us to assess the coupling between CREB activation and neuronal plasticity at the single-cell level. Previously, live imaging revealed increased transcription and translation of the immediate-early genes c-Fos and Arc during experience-dependent plasticity (
      • Barth A.L.
      • Gerkin R.C.
      • Dean K.L.
      Alteration of neuronal firing properties after in vivo experience in a FosGFP transgenic mouse.
      ,
      • Cao V.Y.
      • Ye Y.
      • Mastwal S.
      • Ren M.
      • Coon M.
      • Liu Q.
      • Costa R.M.
      • Wang K.H.
      Motor Learning Consolidates Arc-Expressing Neuronal Ensembles in Secondary Motor Cortex.
      ,
      • Kawashima T.
      • Okuno H.
      • Bito H.
      A new era for functional labeling of neurons: activity-dependent promoters have come of age.
      ,
      • Wang K.H.
      • Majewska A.
      • Schummers J.
      • Farley B.
      • Hu C.
      • Sur M.
      • Tonegawa S.
      In vivo two-photon imaging reveals a role of arc in enhancing orientation specificity in visual cortex.
      ). Although these genes are considered to be regulated by CREB, the temporal coupling between CREB activation and expression of these target genes at the single-cell level is not well understood. Future utilization of simultaneous imaging of CREB activity and immediate early genes expression may help to elucidate experience-dependent transcription-translation interplay.

      Visual Cortical Experience-Dependent Plasticity

      Structural and functional synaptic changes follow sensory deprivation in the primary visual cortex of young and adult mice (
      • Hengen K.B.
      • Lambo M.E.
      • Van Hooser S.D.
      • Katz D.B.
      • Turrigiano G.G.
      Firing rate homeostasis in visual cortex of freely behaving rodents.
      ,
      • Hensch T.K.
      Critical period plasticity in local cortical circuits.
      ,
      • Scholl B.
      • Pattadkal J.J.
      • Priebe N.J.
      Binocular Disparity Selectivity Weakened after Monocular Deprivation in Mouse V1.
      ,
      • Keck T.
      • Keller G.B.
      • Jacobsen R.I.
      • Eysel U.T.
      • Bonhoeffer T.
      • Hübener M.
      Synaptic Scaling and Homeostatic Plasticity in the Mouse Visual Cortex In Vivo.
      ,
      • Rose T.
      • Jaepel J.
      • Hubener M.
      • Bonhoeffer T.
      Cell-specific restoration of stimulus preference after monocular deprivation in the visual cortex.
      ). Although the CREB signaling pathway is thought to be involved in visual cortical plasticity (
      • Mower A.F.
      • Liao D.S.
      • Nestler E.J.
      • Neve R.L.
      • Ramoa A.S.
      cAMP/Ca2+ response element-binding protein function is essential for ocular dominance plasticity.
      ,
      • Pham T.A.
      • Impey S.
      • Storm D.R.
      • Stryker M.P.
      CRE-mediated gene transcription in neocortical neuronal plasticity during the developmental critical period.
      ,
      • Pulimood N.S.
      • Rodrigues W.D.S.
      • Atkinson D.A.
      • Mooney S.M.
      • Medina A.E.
      The Role of CREB, SRF, and MEF2 in Activity-Dependent Neuronal Plasticity in the Visual Cortex.
      ,
      • Suzuki S.
      • al-Noori S.
      • Butt S.A.
      • Pham T.A.
      Regulation of the CREB signaling cascade in the visual cortex by visual experience and neuronal activity.
      ), it has been difficult to correlate differences in CREB signaling within single cells and their activity profiles. Furthermore, although a general relationship between neuronal activity and CREB activity has been described in vitro (
      • Bito H.
      • Deisseroth K.
      • Tsien R.W.
      CREB phosphorylation and dephosphorylation: a Ca(2+)- and stimulus duration-dependent switch for hippocampal gene expression.
      ,
      • Fields R.D.
      • Eshete F.
      • Stevens B.
      • Itoh K.
      Action potential-dependent regulation of gene expression: temporal specificity in ca2+, cAMP-responsive element binding proteins, and mitogen-activated protein kinase signaling.
      ), it has been difficult to precisely determine how this interplay is manifested in cortical circuits in vivo, especially during sensory manipulation.
      Our in vivo imaging approach revealed a dynamic regulation of CREB activity in the visual cortex: DR for 7 days dramatically increases visually evoked CREB activity following minutes and maintains its elevated activity levels for a period of at least 1 day. Interestingly, this increase was not associated with appreciably increased calcium dynamics and, instead, modified the CREB readout or sensitivity to integrated Ca2+ signaling. Other potential mechanisms for this modulation could arise from neuromodulators or neurotrophic brain-derived neurotrophic factor (BDNF)-TrkB signaling, both previously associated with experience-dependent plasticity in the visual cortex (
      • Huang Z.J.
      • Kirkwood A.
      • Pizzorusso T.
      • Porciatti V.
      • Morales B.
      • Bear M.F.
      • Maffei L.
      • Tonegawa S.
      BDNF regulates the maturation of inhibition and the critical period of plasticity in mouse visual cortex.
      ,
      • Yaeger C.E.
      • Ringach D.L.
      • Trachtenberg J.T.
      Neuromodulatory control of localized dendritic spiking in critical period cortex.
      ). Future studies could determine whether this experience-dependent shift represents a general phenomenon of cortical plasticity and identify the underlying mechanisms.
      By establishing an experimental approach to visualize the spatiotemporal dynamics of CREB activity combined with Ca2+ activity in vivo, we correlated functional neuronal responses with molecular signaling dynamics at the microcircuit level. In vivo 2pFLIM of molecular signaling alongside neuronal activity will allow researchers to investigate the mechanisms underlying experience-dependent plasticity within functional neuronal circuits in the brain.

      STAR★Methods

      Key Resources Table

      Tabled 1
      REAGENT or RESOURCESOURCEIDENTIFIER
      Antibodies
      CREB (48H2)Cell signalingCat #9197 RRID-AB_331277
      ATTO647-conjugated RFP-BoosterChromotekCat #rba647n-10
      Bacterial and Virus Strains
      AAV-pSyn-mEGFP-CREB (AAV-9)This studyN/A
      AAV-pSyn-mEGFP-CREB-S133A (AAV-9)This studyN/A
      AAV-pSyn-mCyRFP2-CREB (AAV-9)This studyN/A
      AAV-pSyn-mCherry-KIX-mCherry (AAV-9)This studyN/A
      AAV-pSyn-mMaroon1-KIX-mMaroon1 (AAV-9)This studyN/A
      AAV-pSyn-GCaMP6s
      • Chen T.-W.
      • Wardill T.J.
      • Sun Y.
      • Pulver S.R.
      • Renninger S.L.
      • Baohan A.
      • Schreiter E.R.
      • Kerr R.A.
      • Orger M.B.
      • Jayaraman V.
      • et al.
      Ultrasensitive fluorescent proteins for imaging neuronal activity.
      Addgene_100843
      Chemicals, Peptides, and Recombinant Proteins
      ForskolinTocrisCat #1099
      IBMXTocrisCat #2845
      NKY-80TocrisCat #5071
      Lipofectamine 2000Thermo Fisher ScientificCat # 11668030
      Rimadyl (Carpofen)Zoetis10000319
      DexamethasonePhoenixCat # 24305
      C & B METABONDParkellN/A
      Buprenorphine SRZooPharmN/A
      Experimental Models: Cell Lines
      HEK293 cellsGE Dharmacon, Fischer ScientificCat # HCL4517
      Experimental Models: Organisms/Strains
      Mouse: Swiss WebsterCharles River LaboratoriesCRL:024
      Mouse: C57BL/6JCharles River LaboratoriesCRL:027
      Recombinant DNA
      pCAG-mEGFPThis studyN/A
      pCAG-mEGFP-mCherry
      • Murakoshi H.
      • Lee S.-J.
      • Yasuda R.
      Highly sensitive and quantitative FRET-FLIM imaging in single dendritic spines using improved non-radiative YFP.
      N/A
      pCMV-mEGFP-CREBThis studyN/A
      pCMV-mCyRFP2-CREBThis studyN/A
      pCMV-mCherry-KIX-mCherryThis studyN/A
      pCMV-mMaroon1-KIX-mMaroon1This studyN/A
      pCAG-GCaMP6s
      • Chen T.-W.
      • Wardill T.J.
      • Sun Y.
      • Pulver S.R.
      • Renninger S.L.
      • Baohan A.
      • Schreiter E.R.
      • Kerr R.A.
      • Orger M.B.
      • Jayaraman V.
      • et al.
      Ultrasensitive fluorescent proteins for imaging neuronal activity.
      Addgene_100844
      Software and Algorithms
      Graph Pad Prism 8Graph Padhttps://www.graphpad.com/scientific-software/prism/
      MATLABMathworkshttps://www.mathworks.com/products/matlab.html
      ImageJ
      • Schindelin J.
      • Arganda-Carreras I.
      • Frise E.
      • Kaynig V.
      • Longair M.
      • Pietzsch T.
      • Preibisch S.
      • Rueden C.
      • Saalfeld S.
      • Schmid B.
      • et al.
      Fiji: an open-source platform for biological-image analysis.
      https://imagej.nih.gov/ij/
      Mijihttps://imagej.net/ImageJ
      FLIMageThis studyYasuda lab. https://github.com/ryoheiyasuda/FLIMage_public

      Lead Contact and Materials Availability

      Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Ryohei Yasuda ( Ryohei.yasuda@mpfi.org ).
      Plasmids generated in this study are available from Addgene (Addgene ID 137001-137010).

      Experimental Model and Subject Details

      Mice

      All experimental procedures were approved by the Max Planck Florida Institute for Neuroscience Institutional Animal Care and Use Committee and were performed in accordance with guidelines from the US NIH. For in utero electroporation: Swiss Webster pregnant dams were obtained from Charles River Laboratories and were used in E14.5-15.5. Following electroporation, both male and female offspring mice were used for cranial window surgeries at p45-60. For all other experiments, male C57BL/6 mice were purchased from Charles River Laboratories and used for viral injections and cranial windows surgeries at P45-P80 for in vivo imaging and at p21-28 for acute slice experiments.

      Method Details

      DNA plasmids and AAVs

      For in vivo FLIM characterization, plasmids under CAG promoter were used to express mEGFP, EGFP with A206K mutation (
      • Zacharias D.A.
      • Violin J.D.
      • Newton A.C.
      • Tsien R.Y.
      Partitioning of lipid-modified monomeric GFPs into membrane microdomains of live cells.
      ), and mEGFP-mCherry (
      • Murakoshi H.
      • Lee S.-J.
      • Yasuda R.
      Highly sensitive and quantitative FRET-FLIM imaging in single dendritic spines using improved non-radiative YFP.
      ). For construction of CREB sensor, EGFP followed by a short linker of PTPTPT was fused to the N terminus of CREB (from RSV CREB, a gift from Marc Montminy, Addgene plasmid # 22394). The KIX sequence (amino acids 591-662, generous gift from Oliver Griesbeck) was flanked between mCherry sequences and separated by linkers, as follows: mCherry-SGLRSRA-KIX domain-SAVDGTAGPGSG-mCherry. For cell line experiments, both donor and acceptor were cloned into pEGFP-C1 under the CMV promoter. For expression and production of AAVs, both donor and acceptor were cloned into AAV plasmid backbone under the human synapsin-1 promoter. For red-shifted R-CREB sensor, mEGFP was replaced with mCyRFP2 and mCherry was replaced with mMaroon1 (
      • Bajar B.T.
      • Lam A.J.
      • Badiee R.K.
      • Oh Y.-H.
      • Chu J.
      • Zhou X.X.
      • Kim N.
      • Kim B.B.
      • Chung M.
      • Yablonovitch A.L.
      • et al.
      Fluorescent indicators for simultaneous reporting of all four cell cycle phases.
      ). Mutant CREB donor was assembled by a single point mutation in the CREB reading sequence resulting in S133A mutation in CREB. All cloning procedures were performed using Gibson assembly cloning kits (NEB) and mutations were performed with the Q5 mutagenesis kit (NEB). AAVs for CREB sensor (serotype 9) were packaged in the UNC vector core. AAVs (serotype 9) encoding for hSyn-GCaMP6s (a gift from The Genetically Encoded Neuronal Indicator and Effector Project (GENIE) & Douglas Kim) were purchased from Upen viral core and Addgene.

      Cell culture experiments

      HEK293T cells (GE Dharmacon, Fischer Scientific) were cultured in DMEM supplemented with 10% FBS at 37°C in 5% CO2 and transfected with plasmids using Lipofectamine 2000 (Invitrogen). For G-CREB, mEGFP-CREB/mCh-KIX-mCh were transfected in a ratio of 1:3, and R-CREB, mCyRFP2-CREB/mMaroon1-KIX-mMaroon1 were transfected in a ratio of 1:2. S133 donor mutants were used at same ratios as WT donor. Imaging was performed 24-48 h following transfection. HEK Cells were used as an expression platform only, and were not rigorously tested for potential contamination from other cell lines.

      Organotypic hippocampal slice cultures

      Hippocampal slices were prepared from postnatal 4- to 6-day-old C57BL/6 mice, as described previously (
      • Stoppini L.
      • Buchs P.A.
      • Muller D.
      A simple method for organotypic cultures of nervous tissue.
      ). In brief, 350 μm-thick hippocampal slices were dissected using a tissue chopper. Slices were placed on Millicell membranes (Millipore) in culture medium containing minimal essential medium (Life Technologies), 20% horse serum, 1 mM L-glutamine, 1 mM CaCl2, 2mM MgSO4, 12.9 mM D-glucose, 5.2 mM NaHCO3, 30 mM HEPES, 0.075% ascorbic acid, 1 μg/mL insulin, which was exchanged every other day. Neurons were either transfected via gene gun (
      • Laviv T.
      • Kim B.B.
      • Chu J.
      • Lam A.J.
      • Lin M.Z.
      • Yasuda R.
      Simultaneous dual-color fluorescence lifetime imaging with novel red-shifted fluorescent proteins.
      ) or infected by pressure injection of AAV mixtures using picrospritzer (100-200nl) and imaged at DIV 13-18.

      In utero electroporation

      In utero electroporation was performed as previously reported (
      • Saito T.
      In vivo electroporation in the embryonic mouse central nervous system.
      ). In brief, Swiss Webster E14.5-15.5 timed pregnant dames (Charles River Laboratories) were anesthetized with ∼2% isoflurane and administered 0.1 mg buprenorphine SR (ZooPharm) for analgesia, uterine horns were exposed though an abdominal incision, and the right lateral ventricle of each embryo was injected with plasmids encoding either CAG-mEGFP or CAG-mEGFP-mCherry, at concentration of 1 μg/μl with 0.01% Fast Green dye (Sigma-Aldrich). Five electrical pulses (40 V, 50-ms duration, 1 Hz) were delivered using a NEPA21 electroporator (NEPAGENE) with electrodes directed to the motor cortex.

      AAV injection and cranial window surgeries

      Mice were deeply anesthetized with isoflurane for induction (3%–5%) and maintenance (2%). Mice were administered 1 μg/g buprenorphine SR for analgesia, placed in a stereotaxic frame, and administered 0.2 mg/kg dexamethasone and 5 mg/kg carprofen to prevent edema and inflammation. Following removal of skin and skull exposure, a 2.5-3mm circular craniotomy was performed centered over intended imaging site using a dental drill. The site of injection was determined using stereotactic coordinates, based on mouse brain atlas for the somatosensory (1.4 mm posterior and 3.3mm lateral from bregma), motor (1mm posterior and 1.5 mm lateral from bregma) and visual (0.3 mm anterior and 2.5 mm lateral from lambda) cortex. The pipette was lowered to approximately 300 μm below the pial surface, and AAVs were slowly pressure injected over 5 min using a Picospritzer (Parker) at a rate of 0.3Hz with pulses of 20-100 msec pulse duration. Total volume of injection was 100-350 nl. For G-CREB sensor, we injected a mixture of mEGFP-CREB/mCh-KIX-mCh at 2x1012 and 8x1012 viral genome (vg)/ml. For R-CREB/GCaMP6s we used a mixture of mCyRFP2-CREB, mMaroon-KIX-mMaroon1, and GCaMP6s at concentration of 1x1013, 3x1013, and 1x1012 vg/ml respectively. Following injections, the glass pipette was left in place for 5 minutes and then slowly removed. The skull was sealed using a 2.3 mm, No.1 circular coverglass glued on a 5mm circular coverglass. The coverglass was then cemented to skull along with a head plate to secure the head during imaging using dental cement (C and B Metabond, Parkell). Mice were left to recover and were used for in vivo imaging 21-30 days following surgery. For chronic measurements, mice that showed signs of occlusion of window or tissue damage during the course of imaging experiments were excluded from analysis.

      2pFLIM of organotypic cultures and cell lines

      FLIM imaging of HEK cells and hippocampal slice cultures were performed using custom 2p microscopy as previously described (
      • Laviv T.
      • Kim B.B.
      • Chu J.
      • Lam A.J.
      • Lin M.Z.
      • Yasuda R.
      Simultaneous dual-color fluorescence lifetime imaging with novel red-shifted fluorescent proteins.
      ). Chameleon Ti:sapphire laser (Coherent) was used for excitation at 920 nm. Emission was collected with a 60x 1.0 NA objective (Olympus), divided with a 565-nm dichoic mirror (Chroma) and detected with two PMTs with low transfer time spread (H7422-40p, Hamamatsu) placed after wavelength filters (et520/60-2p for green and et620/60-2p for red, Chroma). PMT voltage was set to 820 V. Average laser intensity for power was set at 1.5–2.0 mW, as measured under the objective. Imaging was performed at room temperature (25°c), except for measurements of kinetics with NMDA and electrical stimulation, which were carried out at 35-36°c. Dual color fluorescence lifetime images were obtained using two time-correlated single-photon counting board (Time Harp 260, Picoquant) controlled with custom software written in C#.

      Slice electrophysiology

      For the characterization of electrophysiological properties of G-CREB expressing cells, C57BL/6 mice (p21-p28) were injected with an AAV mix for G-CREB sensor (mEGFP-CREB and mCh-KIX-mCh) and a mixture of FLEX-CyRFP and Cre on the right and left hemispheres respectively. Following 2-3 weeks of expression, animals were sedated by isoflurane inhalation, and perfused intracardially with ice-cold choline chloride solution (in mM: choline chloride 124, KCl 2.5, NaHCO3 26, MgCl2 3.3, NaH2PO4 1.2, Glucose 10 and CaCl2 0.5; pH 7.4, equilibrated with 95%O2/5%CO2). Brains were then removed and placed in the same chilled choline chloride solution and coronal acute slices of 400μm from left and right hemispheres were collected and placed in oxygenated (95%O2/5%CO2) ACSF (in mM: NaCl 127, KCl 2.5, Glucose 10,NaHCO3 25, NaH2PO4 1.25, MgCl2 2, CaCl2 2) at 32°C for 1h and then maintained at RT for the rest of the experiment. Cortical infected pyramidal neurons were visualized using epifluorescent illumination. Whole cell current clamp recordings were obtained using a Multiclamp 700B amplifier. Patch pipettes (3-6 ΩM) were filled with a K Gluconate solution (in mM: K gluconate 130, Na phosphocreatine 10, MgCl2 4, NaATP 4, MgGTP 0.3, L- Ascorbic acid 3, HEPES 10. pH 7.4, 310 mOsm). Experiments were performed at room temperature (∼23°C) and slices were perfused with oxygenated ACSF. Recordings were digitized at 10 KHz and filtered at 2 KHz. All data was acquired and analyzed with a custom software written in MATLAB.

      Electrical stimulation in organotypic slices

      Following transfection of R-CREB/GCaMP6s, labeled CA1 cells were stimulated by placing a concentric bipolar extracellular electrode near Schaffer collateral axons, with a 350 pulse 10Hz stimuli train. Simultaneous imaging of GCaMP/CREB was performed at 0.78Hz. for on-rate kinetics measurements. Extracellular oxygenated ACSF contained 2mM Ca2+ and 2mM Mg2+ and was maintained at 35-36°c.

      In vivo imaging

      In vivo 2pFLIM was performed using a custom 2-photon microscope, with a Chameleon Ti:sapphire laser (Coherent) tuned to 920-940 nm. The laser was modulated by Pockel cells (Conoptics, Model 350-80LA). Microscope was constructed with one pair of 5mm Galvo mirrors (Cambridge Technologies) coupled with a scan lens (Thorlabs, LSM04-BB) and tube lens (Thorlabs, TTL200-B). Emission was collected with a 20 × 1.0 NA objective (Olympus) or a 16 × 0.8 NA (Nikon), divided with a 565-nm dichroic mirror (Chroma) and detected with two PMTs with low transfer time spread (H7422-40p, Hamamatsu) placed after wavelength filters (et520/60-2p for green and et620/60-2p for red, Chroma). Excitation power was set at 10–40 mW, as measured under the objective. Two color FLIM data was acquired via a Time-Correlated Single Photon Counting board (Time Harp 260, Picoquant) with the temporal resolution of 200 ps using custom software written with C# (source code in https://github.com/ryoheiyasuda/FLIMage_public). For CREB activity imaging in anesthetized mice, images (128 × 128 pixels) were acquired at the frame rate of 7.8 Hz and summed over 600 frames. For dual imaging of CREB activity (R-CREB) and calcium (GCaMP6s) in awake mice, images (128 × 128 pixels) were collected at the frame rate of 7.8Hz and summed over 400 frames. Both R-CREB and GCaMP signals are acquired as fluorescence lifetime images, but GCaMP6s data was analyzed only for intensity.

      Forskolin application during in vivo imaging

      Following 2-3 weeks of cranial window and injection of G-CREB sensor AAV mix, mice were anesthetized with isoflurane and head-fixed with head-plate. A small hole was drilled in the side of the coverglass with a dental drill. Mice were then imaged with 2pFLIM with ACSF solution as immersion fluid, and following 5-10 minutes of baseline imaging, ACSF was replaced with ACSF containing 1mM forskolin, continuing to image same field of view for 30-40 min.

      Immunohistochemistry

      Adult mice were deeply anesthetized by intraperitoneal injection of Ketamine (10 mg/ml)/ Xylazine(1 mg/ml) and intracardially perfused with saline, then 4% paraformaldehyde in 0.1 M phosphate buffer (PB). Whole brains were postfixed for 2 h in the same fixative at 4°C, and PBS overnight. 50 μm thick coronal sections were cut by vibratome (VT1200S, Leica). The sections were permeabilized in 0.5% Triton X-100/ PBS for 10 min at room temperature, and blocked in blocking buffer (4% BSA or 5% normal goat serum (NGS) in 0.1% Triton X-100/ 0.01% NaN3/ PBS). The sections were reacted with primary antibodies at 4°C overnight, incubated with secondary antibodies for 2 h at room temperature, and finally mounted in Fluoromount-G (Southern Biotech). The confocal images were captured with a laser scanning microscope system (LSM880, Zeiss). The following antibodies were used: CREB (48H2) Rabbit mAb (Cell Signaling, #9197), 1:800 in 5% NGS blocking buffer; ATTO647-conjugated RFP-Booster (Chromotek, rba647n-10), 1:600 in 4% BSA blocking buffer; Alexa Fluor 488-conjugated goat anti-rabbit IgG (Invitrogen, A-11034), 1:1000 and Alexa Fluor 405-conjugated goat anti-rabbit IgG (Invitrogen, A-31556), 1:1000 in 5% NGS blocking buffer.

      Enriched environment

      For experiments involving enriched environment, mice were first individually housed in conventional mouse cage, containing only nesting material. To increase sensory enrichment, mice were transferred to a larger size cage which contained various object such as different nesting materials, plastic nests and tunnels, running wheel and metal chains which were hanging from cage wire. Objects were rearranged on a daily basis for a period of 3 days to maintain sensory novelty.

      Dark Rearing experiments

      For dark rearing experiments, 2-3 weeks following viral injection of AAVs encoding R-CREB and GCaMP6s in primary visual cortex1, mice were individually housed in complete darkness in environmental chambers, with air, food and water access. Mice were checked daily with IR illumination and night vision goggles for general health. Following 7 days of dark rearing, mice were transferred in the dark and head-fixed under the microscope for the 1st imaging session. Following 5 minutes of baseline recording of CREB and GCaMP6s activity, mice were exposed to visual stimuli displayed in a monitor situated 15 cm from mice. The stimuli consisted of a short natural movie sequence, lasting 30 s. We measured GCaMP6s responses using the green channel, while measuring R-CREB FLIM signal for 20 s following each movie sequence. This stimulation pattern was repeated for 30 min. In the naive animal group, we performed the same visual stimulation paradigm while housing mice in normal light-dark cycle housing conditions.
      Mice were returned to their respective housing condition, and were imaged 24h later to determine CREB activity levels, which were averaged over a 5 min of imaging session in the dark.

      FLIM analysis

      FLIM analysis was performed with a custom software written in C# (source code: https://github.com/ryoheiyasuda/FLIMage_public). To measure fluorescence lifetime, we fit fluorescence lifetime decay curve F(t) with a monoexponential or biexponential function convolved with the Gaussian pulse response function
      A(t)=A0iPiH(t,t0,τi,τG)


      where Pi is the fractional population with the decay time constant of τi, and H(t) is an exponential function convolved with the Gaussian instrument response function (IRF),
      H(t,t0,ti,tG)=12exp(τG22τi2tt0τi)erfc(τG2τi(tt0)2τiτG),


      in which τG is the width of the Gaussian pulse response function, t0 is the time offset, and erfc is the error function. A0 is the initial fluorescence before convolution. Weighted residuals were calculated as
      E(t)=(F(t)A(t))2/F(t)


      Fitting was performed by minimizing the summed error δ2 = ΣtE(t) for parameters t0, τi (i = 1,2) and τG.
      When measuring binding, instead of numeric subscript, we use subscripts DA and D for values of donor bound to accepter and that of donor alone, respectively. PDA is thus referred to as ‘binding fraction’. To generate a fluorescence lifetime image, we calculated the averaged fluorescence lifetime (τm) by the mean photon arrival time subtracted by t0 in each pixel as
      τm=ttFt/tFtt0.


      We calculated binding fraction in each ROI by fitting fluorescence decay with a function
      A(t,PDA)=A0[(1PDA)H(t,t0,τD,τG)+PDAH(t,t0,τDA,τG)],


      where t0 and τG are obtained from a curve fitting to the fluorescence lifetime decay summed over all ROIs in an image. τD and τDA were obtained from donor alone or CREB sensor expressed in HEK293 cells in a separateexperiment.
      For analysis of CREB activity in vivo, we drew ROIs to positive nuclei in the donor channel and quantified τm and PDA. Cells were chosen for analysis based on photon counting from the cell (> 50,000 photons).
      The theoretical noise of the binding fraction (PDA) due to photon shot noise can be calculated for very short IRF (τG = 0), where τm is approximately:
      τm=(1PDA)τD2+PDAτAD2(1PDA)τD+PDAτAD.
      (Equation 1)


      Since the relationship between the shot-noise of τm (δτm) and photon counting (N) is known to be δτm=τm/N (
      • Yasuda R.
      • Harvey C.D.
      • Zhong H.
      • Sobczyk A.
      • van Aelst L.
      • Svoboda K.
      Supersensitive Ras activation in dendrites and spines revealed by two-photon fluorescence lifetime imaging.
      ), the theoretical noise of the binding fraction (PDA) due to shot noise can be calculated by taking differential of Equation 1 as:
      δPDA=[1PDA(1r2)][1PDA(1r)]r(1r)1N,


      where r = τADD.
      For chronic images of the same cells, imaging field of view was re-identified using blood vessel topography and fluorescence in green/red channels. For anesthetized imaging multiple fields of view were imaged whereas for awake imaging one field of view was imaged and analyzed per animal. For dual GCaMP-CREB imaging, we drew ROIs on cells expressing both GCaMP and CREB and segments of 200-400 frames at 7.8 Hz repetition were summed for FLIM analysis. MATLAB or C# code for FLIM analysis will be provided upon request.

      GCaMP analysis

      Images were corrected for in-plane motion using a correlation-based approach (MATLAB). ROI drawing was performed in ImageJ (
      • Schindelin J.
      • Arganda-Carreras I.
      • Frise E.
      • Kaynig V.
      • Longair M.
      • Pietzsch T.
      • Preibisch S.
      • Rueden C.
      • Saalfeld S.
      • Schmid B.
      • et al.
      Fiji: an open-source platform for biological-image analysis.
      ). ROI’s were circular or drawn using custom software (Cell Magic Wand). Fluorescence time-courses were computed as the mean of all pixels within the ROI at each time point and were extracted using Miji. For each imaging session, fluorescence time courses were computed as changes in fluorescence, ΔF, relative to the baseline fluorescence, F0, which was computed as a 300 point (2.34 s) median filtered fluorescence trace. Fluorescence signals were sometimes contaminated by surrounding neuropil, so we used a computational subtraction procedure to isolate cell calcium signals as follows: (1) perform a robust fit (MATLAB) of ΔF/F0 against neuropil ΔF/F0 (25 pixel radius around ROI center) and (2) subtract a scaled version of the neuropil ΔF/F0, where the scaling factor equals the slope from the robust fit. Cells with a correlation > 0.20 between residual ΔF/F0 and neuropil ΔF/F0 were excluded from further analysis. Summed Ca2+ (∑ΔF/F0) has computed as the sum (across stimulus trials) of the mean ΔF/F0 during visual stimulation.

      Quantification and Statistical Analysis

      Statistical tests of Student’s t test and one or two way ANOVA as indicated in figure legends. GraphPad Prism and MATLAB were used for statistical analysis. Data is presented throughout paper as mean and errors represent SEM, n represents number of cells/animals or cells/slices as described in figure legends. For all statistical tests p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001 and ∗∗∗∗p < 0.001 were considered significant. For comparisons of CREB and CREB-Ca2+ relationships, we used bootstrapped principle components analysis (
      • Sokal R.R.
      • Rohlf F.
      Biometry: the principles and practice of statistics in biological research.
      ). No statistical methods were used to predetermine sample size.

      Data and Code Availability

      The data supporting the current study are available from the corresponding author on request. Code and software for analysis of FLIM data is available on Yasuda lab Github: https://github.com/ryoheiyasuda/FLIMage_public.

      Acknowledgments

      The authors would like to thank to D. Kloetzer for lab management, L. Colgan and C. O’Banion for comments on this manuscript, J. Richards for slice preparation, M. Dowdy for animal care, M. Klement and the MPFI machine shop for technical support, and Dr. K. Padmanabhan for providing the short natural video. This work was supported by National Key Research and Development Program of China grant 2017YFA0700403 , National Natural Science Foundation of China grants 31670872 and 21874145 , and Shenzhen Science and Technology Innovation Committee grant JCYJ20170818164040422 (to J.C.); a long-term post-doctoral fellowship from the Human Frontiers Science Organization (HFSP) (to T.L.); NIH grants DP1NS096787 and R01MH080047 (to R.Y.); Brain Research Foundation grant BRF-SlA-2018-01 (to R.Y.); and the Max Planck Florida Institute for Neuroscience .

      Author Contributions

      T.L. and R.Y. conceived the project. T.L. performed all of the experiments. B.S. contributed to conceptualization and performed Ca2+ and correlation analyses. P.P.B. performed electrophysiological recordings. B.F. assisted with in vitro characterization and animal handling. C.Z. and J.C. developed mCyRFP2. L.Y. provided assistance with microscopy. Y.H. performed immunostaining. T.L., B.S., and R.Y. wrote the paper with feedback from all authors.

      Declaration of Interests

      R.Y. is a founder of Florida Lifetime Imaging LLC.

      Supplemental Information

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