adeno associated virus serotype 9 aav9 vector  (Vector Biolabs)

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: Theranostics

Article Title: Cardiomyocyte-derived OTUD7B promotes cardiac hypertrophy by deubiquitinating SERCA2a

doi: 10.7150/thno.129105

Figure Lengend Snippet: Cardiomyocyte-specific OTUD7B overexpression promotes cardiac hypertrophy by deubiquitinating SERCA2a at K628. (A) Schematic diagram of the TAC-induced mouse model. WT mice were injected with AAV9 cardiomyocyte-specific overexpressing SERCA2a WT or SERCA2a K628R , and OTUD7B OE or empty vector (EV) by the tail vein. After 2 weeks, mice were subjected to TAC. After 4 weeks, the cardiac function of mice was assessed using echocardiography. Mice were then euthanized, and samples were harvested. (B) Representative left ventricular M-mode echocardiographic images. (C-D) Values of ejection fraction (C) and fractional shortening (D). EF, ejection fraction; FS, fractional shortening; n = 6. (E) Representative whole heart images. (F) The ratio of heart weight to body weight (HW/BW). n = 6. (G) The ratio of heart weight to tibial length (HW/TL). n = 6. (H) Representative HE-stained images of cardiac tissue sections. (I-J) Representative images (I) and quantification (J) of wheat germ agglutinin (WGA)-stained cardiac tissue sections (n = 6). (K-L) Representative images (K) and quantification (L) from Masson's trichrome-stained cardiac tissue sections (n = 6). (M) Serum atrial natriuretic peptide (ANP) levels were detected using ELISA kits (n = 6). (N-P) mRNA levels of Myh7 (N), Nppa (O), and Nppb (P) in heart tissues (n = 6). Signal intensities were adjusted using Actb as the normalization control.

Article Snippet: Animals were maintained for 4 weeks after TAC or sham surgery. (3) OTUD7B (OTUD7B OE ) and SERCA2a (SERCA2a WT or SERCA2a K628R ) cardiomyocyte-specific overexpression was achieved using recombinant adeno-associated virus serotype 9 (AAV9) vectors driven by the cTNT promoter (Genechem, Shanghai, China).

Techniques: Over Expression, Injection, Plasmid Preparation, Staining, Enzyme-linked Immunosorbent Assay, Control

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, Clone Assay, Sequencing, Immunofluorescence, 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, 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: 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, 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, Over Expression, Comparison

Journal: General Psychiatry

Article Title: MicroRNA-451a is a candidate biomarker and therapeutic target for major depressive disorder

doi: 10.1136/gpsych-2023-101291

Figure Lengend Snippet: Transgenic overexpression of miR-451a in the mPFC alleviated CRS-induced, depression-like behaviour and neuronal spinal loss in mice. (A) Diagram illustrating the experimental time course for AAV injection (Ad_OE-miR-451a for miR-451a overexpression and Ad_OE-scramble for negative control), CRS model establishment, the behavioural tests and the sacrifice of mice. (B) The representative image of the injected AAV in the mPFC: Cg1, cingulate cortex area 1; Cg2, cingulate cortex area 2; DP, dorsal peduncular cortex; FrA, frontal association cortex; IL, infralimbic cortex; M2, secondary motor cortex; MO, medial orbital cortex; PrL, prelimbic cortex; VO, ventral orbital cortex. (C) Typical fluorescent images of AAV_OE-miR-451a-GFP-infected cells (green) and neurons (red) in the mPFC. (D) The statistical graph shows the percentage of AAV_OE-miR-451a-GFP-infected neurons in total neurons (n=3 mice, 3 slices per mouse). (E) AAV _OE-miR-451a-GFP injection increased miR-451a level in the mPFC (n=4). (F) CRS-induced decrease in sucrose intake preference, which was reversed by prior injection of AAV virus expressing miR-451a (Ad_OE-miR-451a) into the mPFC (Ad_OE-scramble-CON n=17, Ad_OE-miR-451a-CON n=16, Ad_OE-scramble-CRS n=18, Ad_OE-miR-451a-CON n=16). (G) Similarly, overexpression of miR-451a in CRS mice normalised the immobile time in the forced swimming test (Ad_OE-scramble-CON n=17, Ad_OE-miR-451a-CON n=16, Ad_OE-scramble-CRS n=18, Ad_OE-miR-451a-CON n=16). (H) Representative images of dendritic segments and spines of neurons in the mPFC from controls and CRS-exposed mice injected with scrambled and miR-451a overexpression viruses. (I) Quantification revealed that miR-451a overexpression reversed decreases in spine densities of the mPFC neurons of the CRS mice (n=3 per group, 5 neurons per mouse). Data are mean±SEM and analysed by Student’s t-test (E) and two-way ANOVA with Tukey post-hoc test (F, G and I), respectively. AAV, adeno-associated virus serotype 9; ANOVA, analysis of variance; CON, control; CRS, chronic restraint stress; DAPI, diamidino-2-phenylindole; FST, forced swimming test; miR-451a, microRNA-451a; mPFC, medial prefrontal cortex; SEM, standard error of the mean.

Article Snippet: For overexpression of miR-451a in vivo , viral constructs were designed using adeno-associated virus serotype 9 (AAV9) vectors (Genechem, China), encoding a pri-mmu-miR-451a-GFP fusion protein (Ad_OE-miR-451a) or green fluorescent protein (GFP) only as a negative control, under the control of the cytomegalovirus (CMV) promoter (Ad_OE-scramble).

Techniques: Transgenic Assay, Over Expression, Injection, Negative Control, Infection, Virus, Expressing