Journal: iScience

Article Title: Rapid neonatal AAV delivery for adult cortical two-photon imaging of genetically encoded sensors

doi: 10.1016/j.isci.2025.113898

Figure Lengend Snippet: A large PPC neuronal population in a neonatal-AAV-injected mouse stably expresses jGCaMP8f over 4 weeks and exhibits activity that reflects experimental variables (A) Visual (top) and memory (bottom) cue-guided choice tasks. Shading of maze regions and colors of arrows showing correct trajectories for left and right cue trials correspond to (D). (B) Detected jGCaMP8f- and mRuby2-expressing cells in 3 imaging planes across days. (C) Mean projections of layer 2/3 neurons in PPC expressing jGCaMP8f in one imaging plane from sessions on postnatal days 167 and 194. Right: Merged projections after nonrigid alignment. The 10 cells with the highest mean Δ F/F 0 are lettered a-j. Scale bars, 50 μm. (D) Example jGCaMP8f traces from cells with the highest mean Δ F/F 0 during task training. Left column: mean projection of jGCaMP8f activity in a 25-pixel square around each cell during the session on P167 (left) or P194 (right). Upper panels: denoised Δ F/F 0 traces during 8 trials in which the mouse took an efficient path to traverse the stem and made a left or right turn. Shading shows pre-cue distance and reward (sugar water) and punishment (screen flash) periods, corresponding to (A). Colored bars later in discussion traces show whether the cue and correct decision were right (red) or left (blue) for each trial. The bottom panels display the velocity along the Y axis, which is aligned with the stem. (E) Mean Δ F/F 0 in bins of time before the choice time for cells across all 3 imaging planes that were matched between P167 and P194. The Δ F/F 0 scale is normalized to a range of 0–1 for each cell. Cells are sorted according to the time bin with the highest mean Δ F/F 0 on P167. (F) Same as (E) but showing cells unique to P167 and P194. In each plot, cells are sorted according to the time bin with the highest mean Δ F/F 0 on the corresponding day.

Article Snippet: In , AAV9- hSYN -jGCaMP8f (Addgene, 162376, titer ≥ 1×10 13 vg/mL) and AAV9-mDlx-NLS-mRuby2 (Addgene, 99130, titer ≥ 1×10 13 vg/mL) were injected into the left parietal cortex (midway between bregma and lambda, ∼1 mm lateral to midline) of neonatal mice.

Techniques: Injection, Stable Transfection, Activity Assay, Expressing, Imaging

Journal: iScience

Article Title: Rapid neonatal AAV delivery for adult cortical two-photon imaging of genetically encoded sensors

doi: 10.1016/j.isci.2025.113898

Figure Lengend Snippet: Neonatal AAV injection minimizes glial activation as compared to adult AAV injection (A) Photograph of an anesthetized mouse undergoing adult AAV injection. A metal headplate is affixed to the skull, and a burr hole is drilled at the target location. A glass micropipette is then used to gently penetrate the thinnest portion of the burr hole and is lowered to the desired depth. AAV is delivered slowly over a 10–20 min period to ensure precise and controlled injection. (B) Two-photon z-stacks of the dura in adult mice injected with AAVs either neonatally or in adulthood. Scale bars, 100 μm. (C) Dural thickness is strongly anticorrelated with maximum two-photon imaging depth in mice injected with AAV9- hSYN -GCaMP6f as pups (circles) or adults (triangles), with thinner dura associated with deeper imaging (Pearson correlation: r = 0.90, p < 0.001). Right, representative two-photon GCaMP6f images from adult (top; orange triangle from left graph) and pup-injected mice (bottom; green circle from left graph) at their respective cortical depths. Scale bars, 20 μm. (D) Left, coronal sections of the prefrontal cortex of an adult mouse that underwent neonatal AAV9- hSYN- GCaMP6f injection (left hemisphere) followed by adult AAV9- hSYN- GCaMP6f injection (right hemisphere). Sectioned tissue was immunostained against Iba1, GFAP, and DAPI. Scale bars, 1 mm. Right, dashed boxed regions from the left panels highlight increased Iba1 and GFAP immunoreactivity in the adult-injected hemisphere (orange boxed region) compared to the neonatally (green boxed region) injected hemisphere. Scale bars, 100 μm. (E) Average fluorescent intensity measurements of Iba1 and GFAP immunoreactivity across GCaMP6f-expressing regions reveal a significant increase in signal intensity in adult AAV-injected hemisphere mice (orange lines) compared to the pup-injected hemisphere (green lines) (Top: repeated measures aligned rank transform (ART) ANOVA: main effect of injection method, F = 3779.052, p < 0.001, Bottom: repeated measures ART ANOVA: main effect of injection method, F = 3925.961, p < 0.001). Lines represent the mean intensity from 4 animals. (F) Iba1 (left) and GFAP (right) expression indices from individual mice in (E). Pup-injected hemispheres show significantly less Iba1 (paired t test: t = 5.539, p = 0.0116) and GFAP (paired t test: t = 19.97, p = 0.0003) immunoreactivity relative to their contralateral adult-injected hemisphere. See also .

Article Snippet: In , AAV9- hSYN -jGCaMP8f (Addgene, 162376, titer ≥ 1×10 13 vg/mL) and AAV9-mDlx-NLS-mRuby2 (Addgene, 99130, titer ≥ 1×10 13 vg/mL) were injected into the left parietal cortex (midway between bregma and lambda, ∼1 mm lateral to midline) of neonatal mice.

Techniques: Injection, Activation Assay, Imaging, Expressing

Journal: iScience

Article Title: Rapid neonatal AAV delivery for adult cortical two-photon imaging of genetically encoded sensors

doi: 10.1016/j.isci.2025.113898

Figure Lengend Snippet: Chemogenetic modulation of distinct cortical cell types in the prefrontal cortex (A) Timeline of the DREADD-induced modulation of neuronal activity experiment. Transgenic Cre mice or AAV9- CaMKII -Cre were coinjected with both Cre-dependent GCaMP6f and DREADD-hM3D(Gq) as pups at P1, followed by preparation for two-photon imaging at P28. (B) On the day of imaging, layer 2/3 neurons of the PFC were recorded under wakefulness and after CNO injection (orange shaded area). CNO induced the spontaneous activation of these neuronal cell types.

Article Snippet: In , AAV9- hSYN -jGCaMP8f (Addgene, 162376, titer ≥ 1×10 13 vg/mL) and AAV9-mDlx-NLS-mRuby2 (Addgene, 99130, titer ≥ 1×10 13 vg/mL) were injected into the left parietal cortex (midway between bregma and lambda, ∼1 mm lateral to midline) of neonatal mice.

Techniques: Activity Assay, Transgenic Assay, Imaging, Injection, Activation Assay

Journal: iScience

Article Title: Rapid neonatal AAV delivery for adult cortical two-photon imaging of genetically encoded sensors

doi: 10.1016/j.isci.2025.113898

Figure Lengend Snippet: Neonatal pup injection enables the co-expression of four AAV constructs within the same neuronal populations of local cortical microcircuits (A) Two-photon z stack of the prefrontal cortex shows the expression of Cre-dependent reporters (tdTomato, eGFP, and BFP) under control of AAV9- CaMKII -Cre. Scale bars, 50 μm. (B) A representative layer 2/3 imaging plane from the stack at 194 μm depth reveals high overlap of fluorescent signals across pyramidal neurons ( n = 38). Scale bars, 50 μm. (C) Pearson correlation coefficients calculated between tdTomato, eGFP, and BFP fluorescence intensities across individual cells from (B). The heatmap shows the degree of co-expression between each pair of reporters, with warmer colors indicating stronger positive correlations. Notably, eGFP and BFP exhibited a high degree of correlation (r = 0.97), suggesting strong co-expression, while tdTomato showed moderate correlation with BFP (r = 0.65) and lower correlation with eGFP (r = 0.50), indicating some variability in expression levels.

Article Snippet: In , AAV9- hSYN -jGCaMP8f (Addgene, 162376, titer ≥ 1×10 13 vg/mL) and AAV9-mDlx-NLS-mRuby2 (Addgene, 99130, titer ≥ 1×10 13 vg/mL) were injected into the left parietal cortex (midway between bregma and lambda, ∼1 mm lateral to midline) of neonatal mice.

Techniques: Injection, Expressing, Construct, Control, Imaging, Fluorescence