A comparison of viral strategies and model systems to target norepinephrine neurons in the locus coeruleus reveals high variability in transgene expression patterns
Fig 6
PRS×8-mediated constructs to monitor and manipulate LC activity.
(A) PRS×8-jGCaMP8m was injected into one LC of a Dbhcre mouse and hSyn-DIO-jRGECO1a was injected in the contralateral LC. (B) Epifluorescence images of hemi-brain slices expressing PRS×8-driven jGCaMP8m (green, left) and cre-dependent jRGECO1a (magenta, right). Immunostaining against TH is shown in black, while dashed lines indicate the optical fiber tracts. Confocal images of anti-TH and anti-GFP staining, as well as a merged image with quantification of efficiency and specificity are shown next to the overview images. (C) Simultaneous fiber photometry recordings of jGCaMP8m fluorescence (top) and jRGECO1a fluorescence (center), along with the pupil diameter of the mouse (bottom). (D) ΔF (mean ± standard error of the mean) of jGCaMP8m (green) and jRGECO1a (magenta) as well as pupil diameter (gray) locked to local peaks in the derivative of the pupil diameter (indicated by dashed line, n = 171 peaks from 20 min of recording). Only events in the absence of locomotion (gray, bottom) were analyzed. (E) Pupil-aligned functional and isosbestic fluorescence of jGCaMP8m (top) and jRGECO1a (bottom). Functional excitation was done at 470 nm for jGCaMP8m and 560 nm for jRGECO1a, while isosbestic excitation for both indicators was done at 405 nm. (F) ΔF of green-light-filtered GCaMP8m (green) and jRGECO1a (magenta), locked to peaks in the derivative of pupil diameter, exclude major cross-talk from jGCaMP8m to the jRGECO1a channel (n = 52 peaks). (G) ΔF of red-light-filtered jGCaMP8m (green) and jRGECO1a (magenta), locked to peaks in the derivative of pupil diameter, exclude major cross-talk from jRGECO1a to the jGCaMP8m channel (= 84 peaks). (H) Scheme of bilateral injections of PRS×8-ChrimsonR-tdTomato into the LC of a wild-type mouse. (I) Epifluorescence image of a hemi-brain slice expressing PRS×8-driven ChrimsonR-tdTomato (left). tdTomato was amplified using a cross-reacting antibody against mCherry (magenta), while the LC was visualized with an anti-TH staining (black). Dashed lines indicate the fiber position (only the lesion caused by the fiber tip is visible in this slice). Transgene expression was quantified on confocal images (right). (J) Pupil diameter of the mouse during bilateral optogenetic activation of the LC (red bars; 633 nm, ~5 mW, 20 ms pulses at 20 Hz for 4 s). Insets show DeepLabCut-traced videographic images at indicated time points before and after an optogenetic stimulus. (K) Relative pupil diameter (color coded) during a recording session consisting of 30 trials with an inter-trial-interval of 30s. (L) Pupil size in response to optogenetic stimulation (mean ± standard error of the mean across trials shown in K). Absence of locomotion is shown in gray (bottom).