Journal: The EMBO Journal

Article Title: A genetically targeted reporter for PET imaging of deep neuronal circuits in mammalian brains

doi: 10.15252/embj.2021107757

Figure Lengend Snippet: A Schematic illustration of virus construction and marking of neuronal ensemble activation by CNO‐mediated hM3Dq‐DREADD activation. B, C AAVs encoding ecDHFR fused to destabilized Venus (ecDHFR‐d2Venus) with RAM promoter and hM3Dq were co‐introduced into two segregated regions of the somatosensory cortex and imaged under two‐photon microscope. (B) Fluorescent intensity of ecDHFR‐d2Venus in the brain of awake animals was analyzed before (0 min) and after (8 or 24 h) air‐puff mediated whisker stimulation (10 s, five times) or chemogenetic activation of hM3Dq via peripheral administration of CNO (10 mg/kg). Averaged images of 10 stacked frames in serial z position are shown. (C) Averaged ecDHFR‐d2Venus fluorescence intensities, plotted as dF/F0 ratios at different time points after neuronal activation. Data from 6–8 regions / 3–4 mice for each condition are plotted as mean ± SD (red line) and individual regions (blue lines). 8 h vs 24 h, F (2, 21) = 7.715; * P < 0.05 (one‐way ANOVA). D, E Mice expressing ecDHFR‐d2Venus and hM3Dq in a side of hippocampus was sequentially analyzed by [ 18 F]FE‐TMP PET imaging before (left) and 24 h after (right) intraperitoneal CNO injection. To avoid epileptic seizures and immediate death of mice, we used a minimized dose of CNO (0.3 mg/kg) in this experimental condition. About 2 weeks after PET scan, the mice were sacrificed for histochemical analysis. To determine the expression level of the reporter, postmortem brain slice images, as shown in Appendix Fig , were captured by confocal microscope, ROIs were manually placed on the hippocampal region, and averaged fluorescence intensity of ecDHFR‐d2Venus was measured. The background value of non‐infected hippocampal region was set as 1. (D) Representative PET images demonstrate that 0.3 mg/kg CNO‐mediated activation of hM3Dq enhanced the accumulation of radioactive signals in the hippocampus. Averaged images of dynamic scan data at 60–90 min after i.v. injection of [ 18 F]FE‐TMP are shown. Template MRI images were overlaid for spatial alignment. Arrowhead indicates selective accumulation of radioactive signals in the AAV injection site after CNO administration. (E) SUVR (SUV ratio) of ipsilateral hippocampi / reference region (brain stem) signals during dynamic PET scans. Data from ecDHFR‐d2Venus expressing mice ( n = 4) before and after 0.3 mg/kg CNO i.p. injection are plotted. Correlation with R 2 values between SUVR of [ 18 F]FE‐TMP_PET (y‐axis) and relative expression levels of ecDHFR‐d2Venus (x‐axis, fluorescence) in postmortem tissues was determined by linear regression analysis. Source data are available online for this figure.

Article Snippet: AAV encoding hM3Dq was described previously (Nagai et al , ).

Techniques: Virus, Activation Assay, Microscopy, Whisker Assay, Fluorescence, Expressing, Imaging, Injection, Slice Preparation, Infection