Journal: Nature Methods

Article Title: Rapid, biochemical tagging of cellular activity history in vivo

doi: 10.1038/s41592-024-02375-7

Figure Lengend Snippet: A) Schematic of control and experimental conditions. Both wildtype mice not expressing CaST, and wildtype mice injected with CaST in mPFC, were treated with 24 mg/kg biotin + 3 mg/kg psilocybin for 1 hour, and then sacrificed for histology. B) Example FOVs of wildtype mice not expressing CaST (-CaST) or expressing CaST (+CaST) that were injected with biotin and psilocybin. The -CaST control was replicated across two uninjected mice. C) Schematic for using real-time imaging to identify psilocybin-activated neurons in the mPFC. AAV5-CaMKIIa-GCaMP6f was injected into mPFC, and a 1 mm diameter GRIN lens was implanted. 4 weeks later, mice were imaged head-fixed under a 2 P microscope during an IP injection of 5 ml/kg saline or 3 mg/kg psilocybin. D) Background-subtracted mean images of the FOV during a 10-minute saline recording session (left), and a 10-minute psilocybin recording session (right). Active neurons are displayed as warmer pixel colors. Experiment was replicated in two mice. E) Mean 2 P FOV image of the combined saline and psilocybin recordings with all identified neuron masks shown as colored overlays. F) Example Z-scored fluorescence traces of neurons activated by psilocybin (magenta), unaffected by psilocybin (gray), or inhibited by psilocybin (blue), compared to the saline recording. Each recording was 10 minutes long. G) “Psilocybin minus Saline” activity traces were calculated by subtracting the baseline saline Z-scored trace from the psilocybin Z-scored trace for each neuron. The resulting 10-minute-long trace representing the difference is plotted for each neuron in the heatmap to the left, and the average difference for each neuron is plotted as the heatmap to the right labeled “Avg” ( N = 254 cells from 2 mice). Neurons are plotted ranked by the highest to lowest average Z-score difference. The two horizontal bars represent the thresholds for defining “Activated” versus “Inhibited” neurons (>+0.05 = “Activated”, <−0.05 = “Inhibited”). H) The activity traces for the top 50 ranked “Activated” neurons from panel G are shown during the saline and psilocybin recordings (separated by a dashed vertical line). All scale bars, 50 µm.

Article Snippet: For 2P imaging surgeries, 1,000 nl of AAV5-CaMKIIa-GCaMP6f was injected into mPFC (diluted 1:1 in DPBS; Addgene, 100834-AAV5, 2.2 × 10 12 viral genomes per ml titer).

Techniques: Control, Expressing, Injection, Imaging, Microscopy, Saline, Fluorescence, Activity Assay, Labeling

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