Research ArticleHEALTH AND MEDICINE

A transgenic mouse for imaging activity-dependent dynamics of endogenous Arc mRNA in live neurons

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Science Advances  20 Jun 2018:
Vol. 4, no. 6, eaar3448
DOI: 10.1126/sciadv.aar3448
  • Fig. 1 Generation of Arc-PBS knock-in mouse model and behavior characterization.

    (A) Schematic of the endogenous Arc locus, Arc-PBS targeting construct, and the final targeted locus. Blue boxes, homology arms for homologous recombination; gray boxes, UTR; green boxes, Arc coding sequence (Arc-CDS); orange boxes, 24× PBS cassette; black lines, introns; pink boxes, FRT sequences; yellow box, PGK-neomycin resistance gene cassette (PGK-neo). PBS cassette was inserted 250 nucleotides downstream of the stop codon of the Arc gene. (B) Exploratory behavior tested by placing animals in an enriched environment and scored during training. (C) Object recognition memory evaluated by training animals for 5 min and then testing after a long retention interval of 3 hours. Scores indicate preference for the novel object. (D) Spatial memory evaluated by training animals for 5 min and then testing after 45 min of retention. Scores indicate preference for the displaced object. Dashed line shows minimum score required to pass. (E and F) Anxiety-like behavior tested on an elevated plus maze test by scoring percentage of track length traversed in the open tracks (E) and time spent in the center zone in an open-field test (F). n = 8 WT and 10 ArcP/P animals.

  • Fig. 2 PBS tagging does not affect Arc transcription and mRNA stability.

    (A) smFISH probes against Arc-CDS and PBS linkers (PBS) to detect mature full-length PBS-tagged Arc mRNAs. (B) Detection of WT and PBS-tagged allele in WT mouse (top) and heterozygous ArcP/+ mouse (bottom) by smFISH with Arc-CDS and PBS probes in bicuculline (Bic)–stimulated neurons. Inset shows transcription sites. Arrows indicate untagged WT allele; yellow arrowhead indicates PBS-tagged allele. (C and D) Quantification of Arc transcripts produced from WT and PBS-tagged allele, as detected by smFISH in neurons from ArcP/+ mouse after bicuculline stimulation. Graphs represent (C) nascent Arc mRNAs produced at transcription sites at different time points after stimulation (n = 40 neurons for Bic 10′, n = 20 for Bic 30′, n = 15 for Bic 60′; no significant differences, P > 0.05 for all conditions) and (D) mature full-length mRNAs in soma and in dendritic segments (up to 100 μm from soma) in ArcP/+ mouse. Ratios of PBS-tagged mRNAs to total Arc mRNAs are shown in (D). Nonsignificant difference from expected 0.5 value (P = 0.60, n = 49 for soma; P = 0.37, n = 45 for dendrites). (E and F) Detection of full-length Arc mRNAs by two-color smFISH with Arc-CDS and PBS probes in ArcP/P mouse. White arrows indicate PBS-tagged Arc alleles in inset (E), and colocalization of CDS and PBS FISH spots in dendrites (F). (G and H) Correlation between the number of mature cytoplasmic mRNAs detected by Arc-CDS and PBS smFISH in the soma (G) and in the dendrites (H) of ArcP/P neurons, indicated by Pearson’s coefficient (r2) (n = 71 for soma, n = 39 for dendrites). Error bars represent SEM. Scale bars, 5 μm.

  • Fig. 3 Time course of Arc transcription in ArcP/P mouse neurons after stimulation.

    (A) Hippocampal neurons were treated with TTX (1.5 μM) for 12 to 16 hours, followed by release from TTX, and then fixed at different time points for smFISH using probes against the PBS linkers. Transcription from both alleles was observed. (B to D) Graphs representing the time course of average number of nascent transcripts produced at both transcribing alleles (B), average number of mature mRNAs in the soma (C), and the nuclear/cytoplasmic ratio of mRNAs in soma (D). (E) Percentage of Arc-transcribing neurons at different time points following TTX withdrawal (TTX-w) and bicuculline treatment (n = 3 independent experiments, 70 to 80 neurons each time point). (F) Average number of Arc mRNAs in the soma after bicuculline stimulation. Black dashed line indicates average number of mRNAs in the basal condition (n = 40 to 50 neurons per time point). Error bars represent SEM. Scale bar, 5 μm.

  • Fig. 4 Real-time measurements of activity-dependent transcription dynamics of Arc.

    (A) Snapshots of Arc-transcribing alleles, before and after TTX withdrawal (time in minutes). Green and yellow arrowheads indicate transcription from a single and both Arc alleles, respectively. (B) Intensity profiles of transcription sites under basal and stimulated conditions. Solid line indicates the baseline normalized to the nuclear background. Dashed line indicates the threshold for transcriptional burst (ON state). Numbers indicate the count of transcriptional bursts. a.u., arbitrary units. (C) Percentage of time in the actively transcribing state within the 2-hour time window for basal and stimulated conditions (*P < 0.05, two-tailed t test). (D) Inverse cumulative probability distribution of active (ON) duration of Arc transcription fitted to a two-component model, showing two time constants: τ1 and τ2. Percentage of events belonging to either τ1 or τ2 is indicated. CDF, cumulative distribution function. (E) Percentage of events in τ2 duration using different stimulation paradigms. (F) Amplitude of transcriptional burst. (G) Frequency histogram of transcriptional bursts in 2-hour time window. (H) Graph representing the relationship of OFF duration (τOFF) following an ON duration (τON ≥ τ2) in neurons after stimulation. Shaded ellipse indicates means ± 2 SDs (n = 36 alleles). Error bars for all panels indicate SEM (n = 37 cells for basal, 41 for TTX-w, 30 for Bic, and 32 for KCl from three independent experiments). Scale bar, 10 μm.

  • Fig. 5 Dual-color imaging of Ca2+ activity and Arc transcription in live neurons.

    (A) Experimental scheme of measuring both Ca2+ activity and Arc transcription from the same neuron. One green block represents one z scan through the nucleus to detect Arc transcription sites in the green channel. One red block represents continuous imaging of Ca2+ activity at the mid-plane of the nucleus for ~5 min in the red channel. The same neuron was monitored before and after the stimulation by 50 μM bicuculline. (B) Representative Ca2+ image from the red channel. (C) Synchronized Ca2+ spikes measured from the soma (area within dashed line) of cell 1 (top) and cell 2 (bottom) 5 min after bicuculline stimulation. (D) Representative image of Arc transcription in the same neurons as shown in (B). Transcription from both alleles in cell 1 (cyan and magenta arrowheads) versus no transcription site in cell 2. (E) Time course plot of Arc transcription site (TS) intensity in cell 1 (top) and cell 2 (bottom). (F) Representative image of smFISH-IF showing Arc transcription (green) and pCREB (red) in the nucleus [4′,6-diamidino-2-phenylindole (DAPI), blue]. (G) Probability of Arc transcription (TXN) in neurons grouped by pCREB levels in the nucleus. **P < 0.01, *P < 0.05, two-tailed t test. A total of 203 neurons were imaged from four independent experiments. Error bars represent SEM. Scale bars, 5 μm.

  • Fig. 6 Localization and transport dynamics of dendritic Arc mRNA.

    (A) Representative image of Arc mRNAs localized at the neck of a dendritic spine (top right panel) and inside a spine (bottom right panel). (B) Quantification of the average number of mature mRNAs detected in the dendrites (within 100 μm of soma) after TTX withdrawal. (C) Kymograph showing both stationary and actively moving Arc mRNAs in a dendrite. (D) Fraction of mobile Arc mRNAs that moved more than 1.5 μm during 1 min of observation after bicuculline and KCl stimulation. n = 510 and 141 mRNAs for Bic and KCl conditions, respectively. (E) Fraction of mobile Arc mRNAs in the bicuculline washout (control), bicuculline washout + TTX (TTX), and bicuculline washout + TTX + APV + CNQX (TTX + APV + CNQX). n = 510, 427, and 549 mRNAs for control, TTX, and TTX + APV + CNQX conditions, respectively. (F) PDF of the run velocity in control (yellow), TTX (blue), and TTX + APV + CNQX (red) conditions. Velocity of active movement of Arc mRNA was similar among all conditions. (G) Percentage of anterograde (Antero) and retrograde (Retro) movements in all conditions. (H) Histogram of distance traveled by a single run in anterograde and retrograde directions for control, TTX, and TTX + APV + CNQX conditions. *P < 0.05, two-tailed t test. For (E) to (H), a total of 173, 146, and 212 active movements from five independent experiments were analyzed for control, TTX, and TTX + APV + CNQX conditions, respectively. Error bars represent SEM. Scale bars, 5 μm (horizontal) and 10 s (vertical).

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/4/6/eaar3448/DC1

    Supplementary Materials and Methods

    fig. S1. Comparison of homozygous Arc-PBS mouse to WT mouse.

    fig. S2. PCP binding to PBS-tagged mRNAs does not alter mRNA integrity.

    fig. S3. Persistent synchronized Ca2+ spikes after bicuculline washout.

    fig. S4. Synchronized Ca2+ spikes during bicuculline incubation.

    fig. S5. Plot of Ca2+ activity and Arc transcription dynamics.

    fig. S6. Comparison of somatic Ca2+ spikes between neurons with and without Arc transcription within 30 min after bicuculline stimulation.

    table S1. Sequence of the probes used for smFISH against the Arc-CDS and PBS linkers (PBS).

    movie S1. Immediate early activation of transcription from PBS-tagged Arc allele.

    movie S2. Ca2+ activity of neurons induced by bicuculline stimulation.

    movie S3. Arc transcription dynamics induced by bicuculline stimulation.

    movie S4. Dendritic transport of Arc mRNA.

    Reference (61)

  • Supplementary Materials

    This PDF file includes:

    • Supplementary Materials and Methods
    • fig. S1. Comparison of homozygous Arc-PBS mouse to WT mouse.
    • fig. S2. PCP binding to PBS-tagged mRNAs does not alter mRNA integrity.
    • fig. S3. Persistent synchronized Ca2+ spikes after bicuculline washout.
    • fig. S4. Synchronized Ca2+ spikes during bicuculline incubation.
    • fig. S5. Plot of Ca2+ activity and Arc transcription dynamics.
    • fig. S6. Comparison of somatic Ca2+ spikes between neurons with and without Arc transcription within 30 min after bicuculline stimulation.
    • table S1. Sequence of the probes used for smFISH against the Arc-CDS and PBS linkers (PBS).
    • Legends for movies S1 to S4
    • Reference (61)

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    Other Supplementary Material for this manuscript includes the following:

    • movie S1 (.mp4 format). Immediate early activation of transcription from PBS-tagged Arc allele.
    • movie S2 (.mp4 format). Ca2+ activity of neurons induced by bicuculline stimulation.
    • movie S3 (.mp4 format). Arc transcription dynamics induced by bicuculline stimulation.
    • movie S4 (.mp4 format). Dendritic transport of Arc mRNA.

    Files in this Data Supplement:

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