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: 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: 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- CaMK2 -Cre (Addgene, 105558), AAV9- CAG -FLEX-tdTomato (Addgene, 28306), AAV1- CAG -FLEX-eGFP (Addgene, 59331), and AAV9- CAG -FLEX-BFP (Vector Biolabs; 1.06 x 10 13 GC/mL) were targeted to the neonatal prefrontal cortex (∼0.5–1 mm anterior to bregma, 0.5 mm lateral to midline).

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- CaMK2 -Cre (Addgene, 105558), AAV9- CAG -FLEX-tdTomato (Addgene, 28306), AAV1- CAG -FLEX-eGFP (Addgene, 59331), and AAV9- CAG -FLEX-BFP (Vector Biolabs; 1.06 x 10 13 GC/mL) were targeted to the neonatal prefrontal cortex (∼0.5–1 mm anterior to bregma, 0.5 mm lateral to midline).

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- CaMK2 -Cre (Addgene, 105558), AAV9- CAG -FLEX-tdTomato (Addgene, 28306), AAV1- CAG -FLEX-eGFP (Addgene, 59331), and AAV9- CAG -FLEX-BFP (Vector Biolabs; 1.06 x 10 13 GC/mL) were targeted to the neonatal prefrontal cortex (∼0.5–1 mm anterior to bregma, 0.5 mm lateral to midline).

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