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 , DREADD-induced modulation of pyramidal neurons was accomplished with expression of Cre-dependent DREADD-hM 3 D(G q ) (AAV9- hSYN -DIO-hM 3 D(G q )-mCherry; Addgene, 44361) under the human synapsin-1 promoter in Cre-positive mice or mice coexpressing AAV9- CaMK2 -Cre (Addgene, 105558).

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 , DREADD-induced modulation of pyramidal neurons was accomplished with expression of Cre-dependent DREADD-hM 3 D(G q ) (AAV9- hSYN -DIO-hM 3 D(G q )-mCherry; Addgene, 44361) under the human synapsin-1 promoter in Cre-positive mice or mice coexpressing AAV9- CaMK2 -Cre (Addgene, 105558).

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 , DREADD-induced modulation of pyramidal neurons was accomplished with expression of Cre-dependent DREADD-hM 3 D(G q ) (AAV9- hSYN -DIO-hM 3 D(G q )-mCherry; Addgene, 44361) under the human synapsin-1 promoter in Cre-positive mice or mice coexpressing AAV9- CaMK2 -Cre (Addgene, 105558).

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

Journal: Nature Communications

Article Title: Nitrous oxide activates layer 5 prefrontal neurons via SK2 channel inhibition for antidepressant effect

doi: 10.1038/s41467-025-57951-y

Figure Lengend Snippet: a Left, chronic stress was induced with either corticosterone in tap water (CORT) or exposure to screened aggressive male CD-1 mouse (chronic aggressor interactions or CAI). CORT, CAI, and control (exposed to daily handling) mice were subjected to two-photon calcium imaging in the Cg1 (imaging location denoted by green highlighted region with dashed line to cell types) before, during, and after nitrous oxide (N 2 O) exposure. N 2 O was blended, delivered, and monitored under the microscope at 50% for 1 h. Right, CaMK2-expressing excitatory neurons located in layer 2/3 (L2/3) and layer 5 (L5) in CORT mice. Top (room air) and bottom (N 2 O) images show 5 cells from each layer with their fluorescent transients from a 2 min time-series movie collapsed into a single image. Scale bar, 20 μm. Created in BioRender. Cichon, J. (2025) https://BioRender.com/d16f930 . b – d Chronic stress (CORT, N 2 O n = 16, O 2 n = 12; CAI, N 2 O n = 11, O 2 n = 10) increased the time spent immobile in tail suspension test (TST) ( b ) avg. immobility time was 153 ± 7 seconds in CORT and 149 ± 8 s in CAI vs. 86 ± 7 s in controls; Kruskal-Wallis (33): P = 5.6 × 10 −8 followed by Dunn’s multiple comparisons, control vs. CORT: P = 4.1 × 10 −7 , control vs. CAI: P = 6.8 × 10 −6 , decreased exploration of open arms in an elevated plus maze (EPM) ( c ) avg. open arm time was 113 ± 11 s in CORT and 115 ± 16 s in CAI vs. 238 ± 19 seconds in control mice; Kruskal-Wallis (31): P = 1.6 × 10 −7 followed by Dunn’s multiple comparisons, control vs. CORT: P = 1.3 × 10 −6 , control vs. CAI: P = 1.0 x 10 −5 , and reduced sucrose preference index (SPI) ( d ), avg. SPI was 0.06 ± 0.1 in CORT and 0.1 ± 0.1 in CAI vs. 0.5 ± 0.1 in control mice; Kruskal-Wallis (22): P = 1.6 × 10 −7 followed by Dunn’s multiple comparisons, control vs. CORT: P = 2.5 × 10 −5 , control vs. CAI: P = 8.7 × 10 −4 , as compared to control mice (N 2 O n = 14, O 2 n = 14). N 2 O, but not O 2 , therapy rapidly reversed the effects of chronic stress (2-way ANOVA time x treatment: TST CORT, F (2, 78) = 8.1, P = 8.6 x 10 −4 , TST CAI, F (2, 38) = 7.4, P = 0.002; EPM CORT, F (2, 78) = 6.5, P = 0.002, EPM CAI, F (2, 38) = 3.0, P = 0.06; SPI CORT, F (2, 52) = 4.0, P = 0.02, SPI CAI, F (2, 57) = 7.6, P = 0.001). Post treatment comparisons (N 2 O, blue; O 2 , green) are shown in panel ( e ), Representative GCaMP6 traces of the spontaneous activity of individual neurons from L2/3 and L5, shown in ( a ), under wakefulness followed by oxygen (left, green) or N 2 O (right, blue) in control and chronically stressed mice. N 2 O induced the rapid recruitment of L5 neurons as compared to L2/3 across all conditions. f Individual neuronal responses (circles) under room air (yellow) and N 2 O (blue) from all recording regions across Cg1 from control (left) and chronically stressed (middle/CORT and right/CAI) mice. Oxygen plot in Fig. . g L2/3 and L5 population response (colored squares) under room air and N 2 O across control (L2/3: n = 121; L5: n = 102 from 8 mice) and CORT (L2/3: n = 96; L5: n = 122 from 9 mice)/CAI mice (L2/3: n = 61; L5: n = 98 from 4 mice). N 2 O drove the rapid L5 activation across stressed and control mice (Kruskal-Wallis (390): P = 5.3 × 10 −77 followed by Dunn’s multiple comparisons, control: P = 4.8 × 10 −12 , CORT: P = 4.8 × 10 −28 , CAI: P = 3.9 × 10 −33 ) as opposed to L2/3 (Kruskal-Wallis: control/CORT/CAI: P > 0.99). L5 neurons from chronically stressed mice displayed a hypoactivity state relative to control mice (from prior Dunn’s test: CORT, P = 0.006; CAI, P = 0.04). L2/3 activity was also reduced but not significant (CORT, P = 0.80; CAI, P = 0.11). h L5 calcium responses under room air and N 2 O across different genetically defined L5 neuronal subtypes (Kruskal-Wallis (404): P = 4.4 × 10 −85 followed by Dunn’s multiple comparisons, Rbp4 ( n = 89 from 3 mice): P = 1.8 × 10 −17 , Tlx3 ( n = 196 from 4 mice): P = 2.9 × 10 −36 ; Colgalt2 ( n = 149 from 4 mice): P > 0.99). i CaMK2-expressing L5 neuronal responses at different N 2 O concentrations (0, 25, 50, 75% mixed with O 2 ). L5 activating effect observed at 25% with peak effect at 50% (Two-way ANOVA with Bonferroni’s comparisons: 25%, P = 0.03; 50%, P = 4.2 × 10 −7 ; 75%, P = 0.08). j Left, coronal sections of Cg1 from Rbp4-Cre mouse expressing either Cre-dependent chemogenetic variant (tagged with mCherry) hM 3 G q (green) or hM 4 G i (red; Scale bar 100 μm). Right, representative GCaMP6 traces (bottom) and two-photon images (top) of individual L5 neurons recorded during room air and following CNO injection (orange shared area). Scale bar, 20 μm. k CNO-induced Rbp4-L5 neuronal inactivation with hM 4 G i bilaterally in Cg1 ( n = 22) blocked N 2 O’s effect on TST whereas CNO-induced Rbp4-L5 activation with hM 3 G q ( n = 14) masked N 2 O’s effect (two-way ANOVA time x treatment F (4, 166) = 19.8, P = 1.9 x 10 −12 ) post hoc Sidak’s comparisons shown in panel following N 2 O exposure. N 2 O, but not CNO, reduced immobility times in Rbp4 mice expressing tdtTomato ( n = 8). Representative images and traces carried out on at least three animals per group. Error bars show s.e.m.

Article Snippet: Layer 5 (L5) pyramidal neuron and cell type specific interneuron modulation was accomplished with expression of Cre-dependent DREADD-hM 3 D(G q ) (AAV9- hSyn1 -DIO-hM 3 D(G q )-mCherry; Addgene, 44361) or hM 4 D(G i ) (AAV9- hSyn1 -DIO-hM 3 D(G q )-mCherry; Addgene, 44362) under the human synapsin-1 promoter in Cre-positive mice.

Techniques: Control, Imaging, Microscopy, Expressing, Suspension, Activity Assay, Activation Assay, Variant Assay, Injection