Fig. 4: nLightG2 and nLightR2 are suitable to study NE release and neuronal activity via dual-color fiber photometry in vivo.
From: Next-generation multicolor indicators for in vivo imaging of norepinephrine
a, Surgery schematics for sleep experiments. Dbh-iCre mice were injected in the LC with a virus mix to coexpress jGCamp8f and nLightR2 or the mutated (NE-insensitive) nLightR2-ctr. b, Injected mice were implanted with two EEG electrodes (frontal and parietal + a ground over the cerebellum) and two EMG electrodes (for polysomnographic monitoring of sleep–wake behavior) and with an optic fiber over the LC (for dual-color fiber photometry). c, Representative anatomical verification of fiber location (dotted lines, left) and viral expression (middle and right). Middle, micrographs from a mouse co-injected with jGCaMP8f and nLightR2. Right, same for a mouse co-injected with jGCaMP8f and nLightR2-ctr. Zoom-ins show individual colors and overlay. These anatomical verifications were performed for three of seven nLightR2-injected mice and for two of four nLightR2-ctr-injected mice from which the data in d–i were obtained. d, Representative traces of LC dual-color fiber photometry recordings showing jGCaMP8f and nLightR2 signals (z scored) during undisturbed NREM sleep. From the top, vertical bars highlighting scored vigilance states (wake, NREM sleep), time–frequency spectrogram of EEG activity and EMG, nLightR2 (z scored) and jGCaMP8f (z scored) signals. e, As in d for nLightR2-ctr. f, Mean ± s.e.m. for nLightR2 and jGCamp8f z-scored signals around peaks of LC activity during consolidated NREM sleep (1,264 peaks, n = 7 mice). g, As in f for nLightR2-ctr (1,285 peaks, n = 4 mice). h, Swarm plots of peak values (means taken for ±10 s around the LC peak) for nLightR2 (n = 1,264 total detected peaks) and nLightR2-ctr (n = 1,285 total detected peaks) fluorescence; P = 4.0 × 10−51, two-sided Mann–Whitney U-test. i, Swarm plots of cross-correlations between the z-scored nLightR2 or nLightR2-ctr signal with the jGCaMP8f signal around peaks of LC activity (means taken for ±10-s lags around 0-s lag) during consolidated NREM sleep; P = 3.6 × 10−273, two-sided Mann–Whitney U-test with n = 1,264 for nLightR2 and n = 1,285 for nLightR2-ctr. j, Surgery schematics for cued fear conditioning experiments. Wild-type mice were injected in the aBLA with an rAAV mix of PinkyCaMP and nLightG2 or the NE-insensitive nLightG2-ctr. A fiber was implanted to record those indicators simultaneously. k, Same as in c but for PinkyCaMP + nLightG2 or nLightG2-ctr; scale bar, 1,000 µm (overview image) and 20 µm (magnification images). Images were repeated independently ten times with similar results. l, Cued fear conditioning behavioral setting to create an association between an aversive US (1-s-long mild footshock) and a neutral CS (20-s-long tone). m, Heat maps showing the mouse average per trial (rows) of a 65-s interval during CS presentations (ten trials) during baseline and re-exposure sessions for mice coexpressing either PinkyCaMP and nLighG2 (n = 8 mice) or PinkyCaMP and nLighG2-ctr (n = 4 mice). For simplicity, only nLightG2-ctr heat maps are shown. n, Mean PinkyCaMP z-score traces ±s.e.m. (n = 8 mice) of an 85-s interval during CS presentation in baseline and re-exposure sessions. o, Comparison of mean ± s.e.m. z-score nLightG2 (n = 8 mice) and nLightG2-ctr (n = 4 mice) traces in the same time interval and sessions as in n. p, z-Score peaks during CS presentation (average of ten trials per mouse) in both baseline and re-exposure sessions. The aBLA neuronal activity (PinkyCaMP) triggered by tone (CS) increased after memory association (P = 0.0421, two-tailed paired t-test). NE release (nLightG2) triggered by the CS also increased after memory association (P = 6.791 × 10−5, two-tailed paired t-test), whereas no change was seen in the nLightG2-ctr z score across sessions (P = 0.5832, two-tailed paired t-test). q, Same as in m but for the association session. The yellow square represents the US presentation. r, Same as in n but now comparing PinkyCaMP and nLightG2 signals during the association session. s,t, Same as in q and r, respectively, but now comparing PinkyCaMP to nLightG2-ctr signals.
