Journal: Acta Neuropathologica Communications

Article Title: Tau modulation through AAV9 therapy augments Akt/Erk survival signalling in glaucoma mitigating the retinal degenerative phenotype

doi: 10.1186/s40478-024-01804-0

Figure Lengend Snippet: Modulation of Tau expression in C57BL/6 mice retinas A Scheme of AAV plasmid vector containing GFP, mTau, scrambled shRNA, and mTau-shRNAmir sequences. mTau (overexpression) and mTau-shRNAmir (knockdown, KD) sequences were cloned in AAV9 viral vector plasmid fused to the ampicillin-resistance gene. For mTau protein expression, a T2A self-cleaving peptide sequence was used. B Schematic depicting the experimental design and AAV administration experimental timeline. C Immunofluorescence images of retinal sections from control, AAV-GFP, AAV-mTau, AAV-Scramble and AAV-mTau KD groups showing GFP (green, FITC), tau (Green, Alexa Fluor 488), and ptau (Ser199/Ser202) (Red, Cy3). NeuN (red-Cy3) and nucleus (blue, DAPI) (representative images, Scale bar = 50 μm, arrows indicate the changes in the expressions; Antibody concentrations: GFP (1:1000), tau (1:800), ptau (Ser199/Ser202) (1:800), NeuN (1:1000)). D Quantification of GFP relative fluorescence intensity (RFI) percentage (**** P < 0.0001, n = 5). E Quantification of Tau RFI percentage (**** P < 0.0001, n = 5) F Quantification of pTau (Ser199/Ser202) RFI percentage (**** P < 0.0001, n = 5). Statistical significance was assessed by employing One-way ANOVA analysis with Tukey’s multiple comparison test

Article Snippet: For the over expression of the Tau, murine mutant Tau (P232S) cDNA (NCBI ref: BC014748) under the transcriptional control of the cytomegalovirus chicken β-actin (CAG2) hybrid promotor was inserted into the adeno-associated virus serotype 9 (AAV9) vector with the enhanced green fluorescence protein reporter (eGFP) and 2A linker in between GFP and the mTau sequence (AAV9-CAG2-eGFP-2A-mTau (P232S)-WPRE or AAV9- Tau) (Vector Biolabs, USA).

Techniques: Expressing, Plasmid Preparation, shRNA, Over Expression, Knockdown, Clone Assay, Sequencing, Immunofluorescence, Control, Fluorescence, Comparison

Journal: Acta Neuropathologica Communications

Article Title: Tau modulation through AAV9 therapy augments Akt/Erk survival signalling in glaucoma mitigating the retinal degenerative phenotype

doi: 10.1186/s40478-024-01804-0

Figure Lengend Snippet: Electrophysiological and structural alterations in C57BL/6 mice retinas subjected to AAV-tau overexpression and AAV-tau knockdown under normal IOP conditions. A Positive scotopic threshold responses (pSTRs) of the control, AAV-GFP, and AAV-mTau injected mice. B Quantification of amplitudes indicates a significant decrease in pSTR amplitudes in AAV9-Tau overexpressing mice (n = 10, P < 0.0001 t-test). C pSTRs of the control, AAV-scrambled, and AAV-tau shRNA expressing mice and D their quantification indicates a significant decrease of pSTR amplitudes in AAV9-KD mice retinas (n = 10, **** P < 0.0001, t-test). No significant difference in pSTR amplitudes was observed between the control and AAV9-GFP or AAV9-scramble-shRNA sequence-expressing eyes. E H and E staining of retinal sections shows GCL cell density loss in Tau overexpression and Tau KD retinas compared to the controls, arrows indicate the changes in cell densities (representative images, Scale bar = 50 μm). F Quantification of cell density in the GCL from the retinal H and E-stained images (**** P < 0.0001, One-way ANOVA analysis with Tukey’s multiple comparison test, n = 5 for each group)

Article Snippet: For the over expression of the Tau, murine mutant Tau (P232S) cDNA (NCBI ref: BC014748) under the transcriptional control of the cytomegalovirus chicken β-actin (CAG2) hybrid promotor was inserted into the adeno-associated virus serotype 9 (AAV9) vector with the enhanced green fluorescence protein reporter (eGFP) and 2A linker in between GFP and the mTau sequence (AAV9-CAG2-eGFP-2A-mTau (P232S)-WPRE or AAV9- Tau) (Vector Biolabs, USA).

Techniques: Over Expression, Knockdown, Control, Injection, shRNA, Expressing, Sequencing, Staining, Comparison

Journal: Acta Neuropathologica Communications

Article Title: Tau modulation through AAV9 therapy augments Akt/Erk survival signalling in glaucoma mitigating the retinal degenerative phenotype

doi: 10.1186/s40478-024-01804-0

Figure Lengend Snippet: Tau overexpressing mice retinas shows exacerbated degenerative changes in the inner retina in high-IOP glaucoma condition. A Chronic elevation of IOP in C57BL/6 mice eyes was induced by intracameral microbead injections in control, AAV-GFP and AAV-mTau administered mice for 2 months. B Positive scotopic threshold response (pSTR) traces from control, glaucoma, glaucoma + AAV-GFP, and glaucoma + AAV-mTau overexpression mice retinas, as indicated and C their pSTR amplitudes quantification indicated a significant decline of the pSTR in the glaucoma AAV9-tau overexpression animals. (n = 10, *** P < 0.001 **** P < 0.0001) D Western blot analysis of Tau, pTau S199/202 and pTau S404 levels in control and glaucomatous retina. β-actin was used as a loading control E Fold change of Tau, pTau S199/202 and pTau. S404 showed a significant increase in retinas in high IOP conditions (*** P < 0.001, n = 3 each group, t-test). F H and E staining of retinal sections of control, glaucoma, glaucoma + AAV9-GFP, and glaucoma + AAV9-mTau mice (representative images, arrows indicate the changes in cell densities, Scale bar, 50 μm). G Quantification of the cell density in GCL indicated a significant decrease in the number of cells in glaucoma and AAV-tau overexpressed retinas compared to the controls (n = 5, **** P < 0.0001, One-way ANOVA analysis with Tukey’s multiple comparison test)

Article Snippet: For the over expression of the Tau, murine mutant Tau (P232S) cDNA (NCBI ref: BC014748) under the transcriptional control of the cytomegalovirus chicken β-actin (CAG2) hybrid promotor was inserted into the adeno-associated virus serotype 9 (AAV9) vector with the enhanced green fluorescence protein reporter (eGFP) and 2A linker in between GFP and the mTau sequence (AAV9-CAG2-eGFP-2A-mTau (P232S)-WPRE or AAV9- Tau) (Vector Biolabs, USA).

Techniques: Control, Over Expression, Western Blot, Staining, Comparison

Journal: Acta Neuropathologica Communications

Article Title: Tau modulation through AAV9 therapy augments Akt/Erk survival signalling in glaucoma mitigating the retinal degenerative phenotype

doi: 10.1186/s40478-024-01804-0

Figure Lengend Snippet: Silencing of Tau expression using AAV protects the inner retinal function and laminar structure in glaucomatous eyes. A Chronic elevation of IOP in C57BL/6 mice eyes was induced by intracameral microbead injections for 2 months. B Positive scotopic threshold response (pSTR) traces from control, glaucoma, glaucoma + scrambled and glaucoma + Tau KD mice retinas, as indicated. C Quantification of the pSTR amplitudes from the glaucoma AAV9-Tau KD mice eyes compared to glaucoma scrambled sequence expressing controls indicated significant protection against pSTR amplitudes loss in the glaucoma-AAV-Tau KD animals. (n = 10, *** P < 0.001, One-way ANOVA analysis with Tukey’s multiple comparison test). D H and E staining of retinal sections of the control, glaucoma, glaucoma + AAV9-scramble, and glaucoma + AAV9-tau KD mice eyes. (representative images, arrows indicate the changes in cell densities, Scale bar, 50 μm). E Quantification of the cell density in GCL indicated significant protection against cell loss in glaucoma AAV9-tau KD-subjected retinas compared to the microbead-injected control eyes. (n = 5, *** P < 0.001, **** P < 0.0001, One-way ANOVA analysis with Tukey’s multiple comparison test)

Article Snippet: For the over expression of the Tau, murine mutant Tau (P232S) cDNA (NCBI ref: BC014748) under the transcriptional control of the cytomegalovirus chicken β-actin (CAG2) hybrid promotor was inserted into the adeno-associated virus serotype 9 (AAV9) vector with the enhanced green fluorescence protein reporter (eGFP) and 2A linker in between GFP and the mTau sequence (AAV9-CAG2-eGFP-2A-mTau (P232S)-WPRE or AAV9- Tau) (Vector Biolabs, USA).

Techniques: Expressing, Control, Sequencing, Comparison, Staining, Injection

Journal: Acta Neuropathologica Communications

Article Title: Tau modulation through AAV9 therapy augments Akt/Erk survival signalling in glaucoma mitigating the retinal degenerative phenotype

doi: 10.1186/s40478-024-01804-0

Figure Lengend Snippet: Western blot (WB) analysis of retinal endoplasmic reticulum (ER) stress markers changes in glaucoma and Tau modulation conditions. A WBs of control and glaucoma C57BL/6 mice retinal lysates probed with GRP78(1:1000), CHOP(1:1000), P-PERK(1:1000), and β-actin (1:5000) antibodies and B-D their respective relative band intensities quantified as the fold change using β-actin as loading control (n = 3 **** P < 0.0001, t-test). E WB of retinal lysates of the control, glaucoma, AAV-GFP, AAV-mTau (overexpression), AAV-GFP + glaucoma, and AAV-mTau + glaucoma mice were probed with GRP-78, CHOP, P-PERK, and actin antibodies, and F-H their respective relative band intensities quantified as the fold change using β-actin as loading control (n = 3, ** P < 0.01, *** P < 0.001, **** P < 0.0001, One-way ANOVA analysis with Tukey’s multiple comparison test,). I WB of retinal lysates from control, glaucoma, AAV9-scramble, AAV-mTau KD, AAV9-scramble + glaucoma, AAV-mTau KD + glaucoma mice were probed with GRP 78, CHOP, P-PERK, and actin antibodies, and (J-L) their respective band intensities quantified as fold change using β-actin as a loading control (*** P < 0.001, **** P < 0.0001, One-way ANOVA analysis with Tukey’s multiple comparison test, n = 3 per group)

Article Snippet: For the over expression of the Tau, murine mutant Tau (P232S) cDNA (NCBI ref: BC014748) under the transcriptional control of the cytomegalovirus chicken β-actin (CAG2) hybrid promotor was inserted into the adeno-associated virus serotype 9 (AAV9) vector with the enhanced green fluorescence protein reporter (eGFP) and 2A linker in between GFP and the mTau sequence (AAV9-CAG2-eGFP-2A-mTau (P232S)-WPRE or AAV9- Tau) (Vector Biolabs, USA).

Techniques: Western Blot, Control, Over Expression, Comparison

Journal: bioRxiv

Article Title: Reversible synaptic deficits in early-stage Batten disease

doi: 10.1101/2025.11.12.687979

Figure Lengend Snippet: Subtle changes in intrinsic neuronal excitability in Cln3 Δex7/8 mice. (a, b) Representative voltage responses from DG-GC to 300 ms current pulses: - 50 pA to 50 pA current steps in 10 pA increments and voltage-current relations (4-months: left, n= 20,32; N= 3,3 [WT, Cln3 Δex7/8 ]; 3-weeks: right, n= 38,33; N= 5,4 [WT, Cln3 Δex7/8 ]). (c, d) Firing pattern of DG-GCs in response to 1 second sub-threshold and hyperpolarizing current pulses: number of action potentials (AP) plotted against increasing current steps (4-months: left, n= 26,30; N= 4,5 [WT, Cln3 Δex7/8 ]; 3-weeks: right, n= 27,24; N= 5,4 [WT, Cln3 Δex7/8 ]). Data are mean ± SEM, **p < 0.01, *p < 0.05; Mann-Whitney test

Article Snippet: For presynaptic rescue experiments, a 400 nL cocktail of AAV9-hSyn-h CLN3 -IRES-EGFP (Packgene) and AAV9-hSyn-hChR2(H134R)-mCherry (Addgene # 26976-AAV9; deposited by Karl Deisseroth), containing 2.5 × 10 genome copies of each virus, was bilaterally injected into the entorhinal cortex (AP = - 5.2 mm; ML = ±3.4 mm; DV = - 3.5 mm).

Techniques: MANN-WHITNEY

Journal: bioRxiv

Article Title: Reversible synaptic deficits in early-stage Batten disease

doi: 10.1101/2025.11.12.687979

Figure Lengend Snippet: Impaired synaptic transmission in Cln3 Δex7/8 mice. (a, b) Representative mEPSC traces from DG-GCs with summary bar plots of frequency and amplitude (4-months: left, n= 17,26; N= 3,3 [WT, Cln3 Δex7/8 ]; 3-weeks: right, n= 28,31; N= 3,3 [WT, Cln3 Δex7/8 ]). (c, d) Representative mIPSC traces from DG-GCs with summary bar plots of frequency and amplitude (4-months: left, n= 31,40; N= 5,5 [WT, Cln3 Δex7/8 ]; 3-weeks: right, n= 12,13; N= 2,3 [WT, Cln3 Δex7/8 ]). Data are median [IQR]; ns, non-significant; p-values from Mann-Whitney tests

Article Snippet: For presynaptic rescue experiments, a 400 nL cocktail of AAV9-hSyn-h CLN3 -IRES-EGFP (Packgene) and AAV9-hSyn-hChR2(H134R)-mCherry (Addgene # 26976-AAV9; deposited by Karl Deisseroth), containing 2.5 × 10 genome copies of each virus, was bilaterally injected into the entorhinal cortex (AP = - 5.2 mm; ML = ±3.4 mm; DV = - 3.5 mm).

Techniques: Transmission Assay, MANN-WHITNEY

Journal: bioRxiv

Article Title: Reversible synaptic deficits in early-stage Batten disease

doi: 10.1101/2025.11.12.687979

Figure Lengend Snippet: Loss of Cln3 impairs synaptic strength in Cln3 Δex7/8 mice. (a) Schematic of perforant pathway stimulation (Rec., recording pipette; Stim., stimulation electrode). (b) Example traces of evoked AMPA- and NMDA-receptor-mediated currents at -70 mV and +40 mV, respectively; stimulus artifacts removed (replaced by a black line) and summary bar plot of AMPA/NMDA ratio (n= 33,42; N= 5,6 [WT, Cln3 Δex7/8 ]). (c) Representative traces of evoked NMDA receptor- mediated currents at +40 mV and peak amplitudes of NMDAR-mediated currents plotted against increasing stimulus intensities (μA). (d) Summary bar plot of decay time constant (τ) for NMDA receptor-mediated currents (n= 22,30; N= 2,2 [WT, Cln3 Δex7/8 ]). Data are mean [95% CI]; ns, non-significant; p-values from Unpaired t-tests

Article Snippet: For presynaptic rescue experiments, a 400 nL cocktail of AAV9-hSyn-h CLN3 -IRES-EGFP (Packgene) and AAV9-hSyn-hChR2(H134R)-mCherry (Addgene # 26976-AAV9; deposited by Karl Deisseroth), containing 2.5 × 10 genome copies of each virus, was bilaterally injected into the entorhinal cortex (AP = - 5.2 mm; ML = ±3.4 mm; DV = - 3.5 mm).

Techniques: Transferring

Journal: bioRxiv

Article Title: Reversible synaptic deficits in early-stage Batten disease

doi: 10.1101/2025.11.12.687979

Figure Lengend Snippet: Hippocampal neurons of Cln3 Δex7/8 mice have reduced spine number and dendritic complexity. (a) Confocal images of dendritic spines in the medial molecular layer of DG (inset scale bar, 1 μm). (b) Representative dendritic arbors of DG granule cells from WT and Cln3 Δex7/8 . Left: confocal images with dendrites visualized in green. Right: corresponding reconstructed tracings used for Sholl analysis. Scale bar: 50 μm. (c) Summary bar plots of total spine density and (d) spine subtype counts per 10 μm of dendritic length (n= 43,39; N= 8,5 [WT, Cln3 Δex7/8 ]). (e) Sholl analysis quantifying dendritic intersections and (f) summary bar plot of total dendritic length (n= 17,26; N= 3,5 [WT, Cln3 Δex7/8 ]). Data are mean [95% CI] in (c) and median [IQR] in (d, f); ns, non-significant; p-values from Unpaired t-test (total spine density) and Mann-Whitney tests with multiple-comparison FDR correction (spine subtype)

Article Snippet: For presynaptic rescue experiments, a 400 nL cocktail of AAV9-hSyn-h CLN3 -IRES-EGFP (Packgene) and AAV9-hSyn-hChR2(H134R)-mCherry (Addgene # 26976-AAV9; deposited by Karl Deisseroth), containing 2.5 × 10 genome copies of each virus, was bilaterally injected into the entorhinal cortex (AP = - 5.2 mm; ML = ±3.4 mm; DV = - 3.5 mm).

Techniques: MANN-WHITNEY, Comparison

Journal: bioRxiv

Article Title: Reversible synaptic deficits in early-stage Batten disease

doi: 10.1101/2025.11.12.687979

Figure Lengend Snippet: Synapses in Cln3 Δex7/8 mice have reduced vesicle release probability. (a) Representative trace of evoked EPSCs in response to 30 Hz perforant pathway stimulation (black vertical lines above the traces indicate stimulus times). (b) Cumulative EPSC plot with linear fit (black line) of the last 6 points (steady state). (c) Summary bar plots of release probability (P r ), readily releasable pool (RRP) size, and (d) slope (recovery rate) (n= 31,25; N= 4,3 [WT, Cln3 Δex7/8 ]). Data are mean [95% CI] for P r and median [IQR] for RRP size and slope; ns, non-significant; p-values from Unpaired t-test (P r ) and Mann-Whitney tests (RRP size, slope)

Article Snippet: For presynaptic rescue experiments, a 400 nL cocktail of AAV9-hSyn-h CLN3 -IRES-EGFP (Packgene) and AAV9-hSyn-hChR2(H134R)-mCherry (Addgene # 26976-AAV9; deposited by Karl Deisseroth), containing 2.5 × 10 genome copies of each virus, was bilaterally injected into the entorhinal cortex (AP = - 5.2 mm; ML = ±3.4 mm; DV = - 3.5 mm).

Techniques: MANN-WHITNEY

Journal: bioRxiv

Article Title: Reversible synaptic deficits in early-stage Batten disease

doi: 10.1101/2025.11.12.687979

Figure Lengend Snippet: Hippocampal long-term potentiation (LTP) is intact in Cln3 Δex7/8 mice. (a) Schematic of LTP induction in the DG via stimulation of the perforant pathway (MPP, medial perforant pathway, induced; LPP, lateral perforant pathway, control). (b) Example traces of evoked synaptic currents at baseline (grey) and after LTP induction for WT and Cln3 (colored). (c) Time course of normalized EPSC peaks showing LTP induced at the MPP-GC synapse at time 0 (black arrow), while LPP synapses show heterosynaptic depression. Data are mean ± SEM. (d) Summary bar plots of induced and control EPSC peaks normalized to baseline for the first and last 10 minutes (n= 13,14; N= 6,5 [WT, Cln3 Δex7/8 ]). Data are mean [95% CI]; ns, non-significant; two-way RM ANOVA with Sidak post hoc test

Article Snippet: For presynaptic rescue experiments, a 400 nL cocktail of AAV9-hSyn-h CLN3 -IRES-EGFP (Packgene) and AAV9-hSyn-hChR2(H134R)-mCherry (Addgene # 26976-AAV9; deposited by Karl Deisseroth), containing 2.5 × 10 genome copies of each virus, was bilaterally injected into the entorhinal cortex (AP = - 5.2 mm; ML = ±3.4 mm; DV = - 3.5 mm).

Techniques: Control

Journal: bioRxiv

Article Title: Reversible synaptic deficits in early-stage Batten disease

doi: 10.1101/2025.11.12.687979

Figure Lengend Snippet: Re-expression of CLN3 in the presynaptic neurons of Cln3 Δex7/8 mice improves defective synaptic release. (a) Schematic of AAV injections into the entorhinal cortex (EC) to re-introduce CLN3 in presynaptic neurons, combined with optogenetics for selective activation of CLN3 -expressing perforant pathway axons. (b) Representative fluorescence images showing CLN3-EGFP and ChR2-mCherry expression in the EC and perforant pathway; merged image shows their colocalization in the molecular layer (ML) of DG (inset, scale bar: 100 µm). Sub, subiculum; GCL, granule cell layer. (c) Representative trace of evoked EPSCs in response to 30 Hz optical stimulation of the perforant pathway (blue vertical lines above the traces mark stimulus times). The eEPSC traces in the dotted box shows a zoomed-in portion of the high-frequency optical stimulation period. (d) Cumulative EPSC plot with linear fit (black line) of the last 10 points (steady state). (e) Summary bar plots of release probability (P r ), readily releasable pool (RRP) size, and slope (n= 22,20,26; N= 3,3,4 [WT, Cln3 Δex7/8 , Cln3 Δex7/8 + AAV9. CLN3 ]). Data are median [IQR]; ns, non-significant; p-values from Kruskal-Wallis test followed by Dunn’s multiple comparisons test

Article Snippet: For presynaptic rescue experiments, a 400 nL cocktail of AAV9-hSyn-h CLN3 -IRES-EGFP (Packgene) and AAV9-hSyn-hChR2(H134R)-mCherry (Addgene # 26976-AAV9; deposited by Karl Deisseroth), containing 2.5 × 10 genome copies of each virus, was bilaterally injected into the entorhinal cortex (AP = - 5.2 mm; ML = ±3.4 mm; DV = - 3.5 mm).

Techniques: Expressing, Introduce, Optogenetics, Activation Assay, Fluorescence

Journal: bioRxiv

Article Title: Reversible synaptic deficits in early-stage Batten disease

doi: 10.1101/2025.11.12.687979

Figure Lengend Snippet: Postsynaptic re-expression of CLN3 normalizes synaptic strength in Cln3 Δex7/8 mice. (a) Schematic of AAV injections into the dentate gyrus to re-introduce CLN3 in postsynaptic DG granule cells. (b) DIC-IR image showing CLN3-EGFP expression and positions of stimulation (Stim. E) and recording (rec. GFP+) electrodes during electrophysiological recordings. (c) Post hoc verification of patched cells by immunostaining after biocytin filling via patch pipette; merged image shows colocalization of CLN3-EGFP and biocytin, confirming successful targeting. (d) Example traces of evoked AMPA and NMDA receptor-mediated currents at holding potentials of -70 mV and +40 mV, respectively. Stimulus artifacts are removed for clarity (replaced with black vertical line). (e) Summary bar plots of AMPA/NMDA ratio (n= 33,45; N= 5,5 [ Cln3 Δex7/8 , Cln3 Δex7/8 + AAV9. CLN3 ]) and (f) mEPSC frequency and amplitude (n= 25, 22; N= 4,3 [ Cln3 Δex7/8 , Cln3 Δex7/8 + AAV9. CLN3 ]). Horizontal dashed line in blue above the data bars indicate WT values. Data are median [IQR] for AN ratio and mEPSC frequency and mean [95% CI] for mEPSC amplitude; ns, non-significant; p-values from Mann-Whitney tests and Unpaired t-test

Article Snippet: For presynaptic rescue experiments, a 400 nL cocktail of AAV9-hSyn-h CLN3 -IRES-EGFP (Packgene) and AAV9-hSyn-hChR2(H134R)-mCherry (Addgene # 26976-AAV9; deposited by Karl Deisseroth), containing 2.5 × 10 genome copies of each virus, was bilaterally injected into the entorhinal cortex (AP = - 5.2 mm; ML = ±3.4 mm; DV = - 3.5 mm).

Techniques: Expressing, Introduce, Immunostaining, Transferring, MANN-WHITNEY

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

Journal: iScience

Article Title: Astrocytic FABP5 mediates retrograde endocannabinoid transport at central synapses

doi: 10.1016/j.isci.2025.112342

Figure Lengend Snippet:

Article Snippet: The following AAVs were used: AAV-CAG-FABP5 expressing mouse FABP5 under the CAG promoter (Duke VVC #pBK366, serotype AAV9), AAV-CAG-FABP5 MUT expressing FABP5 MUT (Duke VVC #1937, serotype AAV9), AAV-CAG-FABP5 SEC expressing FABP5 SEC (Duke VVC #pBK383, serotype AAV9), AAV-CAG-FABP7 expressing mouse FABP7 (Duke VVC #pBK1433, serotype AAV9), AAV-CMV-Cre (Addgene, #105545, serotype AAV9), AAV-CMV-GFP (Addgene, #105530, serotype AAV9), AAV-hSyn-Cre (Addgene, #105540, serotype AAV5), pAAV-hSyn-EGFP (Addgene, #50465, serotype AAV5), AAV-gfaABC1D-FABP5 expressing FABP5 under the astrocyte-specific gfaABC1D promoter (Duke VVC #pBK1762, serotype AAV8), pAAV.GFAP.eGFP.WPRE.hGH (Addgene, #105549, serotype AAV5), and AAV-hSyn-FABP5 (Duke VVC #pBK1761, serotype AAV5).

Techniques: Virus, Recombinant, Software, Imaging, Transfection, Real-time Polymerase Chain Reaction, SYBR Green Assay, Mass Spectrometry, Control, Microscopy