aav dio hm4di mcherry  (Addgene inc)

Journal: Nature Communications

Article Title: Stabilization of V1 interneuron-motor neuron connectivity ameliorates motor phenotype in a mouse model of ALS

doi: 10.1038/s41467-024-48925-7

Figure Lengend Snippet: a Intraspinal injections were performed in WT, En1 cre , SOD1 G93A and SOD1 G93A ;En1 cre mice, all genotypes were injected in L1-L3 spinal segments. b Schematic of cre-dependent constructs designed to overexpress ESYT1 and mCherry . c – d Tile reconstruction of a longitudinal section of the spinal cord of an En1 cre mouse upon overexpression of the AAV8-hSYN-DIO-mCherry virus validates successful cre-dependent expression in the ventral/medial areas of the cord. AAV8-hSYN-DIO-hESYT1-W3SL driven overexpression was analyzed by RNAscope utilizing a probe recognizing the inverted viral construct upon cre-recombination, e Expression of viral-h ESYT1 in En1 cre mice. f Close-up microphotograph showing a neuron overexpressing ESYT1 . g The same neuron is also positive for En1 transcript. h ESYT1 was not detected in WT injected mice. i , j En1 positive neuron negative for ESYT1 . k Quantification of ESYT1 positive neurons in the lumbar spinal cord of En1 cre and WT mice, 76% of En1 + neurons are positive for ESYT1 ( t -test, two-tailed, P = 0.0052, En1 cre N = 5, WT N = 3). l – w Esyt1 protein expression in interneurons found within the ventral horn of the spinal cord in the four experimental conditions. l – n Esyt1 expression in WT mice; ( o – q ) Esyt1 overexpression in En1 cre healthy mice; ( r – t ) Esyt1 is downregulated in the SOD1 G93A mice and ( u – w ) expression is rescued after AAV8-hSyn-DIO-hEsyt1-W3SL delivery in SOD1 G93A ;En1 cre mice. x Quantifications of Esyt1 intensities in interneurons found within the ventral horn of the spinal cord at P112 in all four experimental conditions show increased Esyt1 protein expression in the SOD1 G93A ;En1 cre mice (One way ANOVA and Fisher’s LSD post hoc P = 0.003; WT = 3, En1 cre N = 2, SOD1 G93A N = 4, SOD1 G93A ;En1 cre N = 3; 10–20 interneurons analyzed per condition). Scale bar in c = 100 μm, in d = 50 μm, in e , h = 20 μm and in l , o , r , u = 20 μm. All graphs show mean values ± SEM. Source data are provided as a Source Data file.

Article Snippet: The AAV overexpressing ESYT1 was developed utilizing a pAAV-hSYN-DIO[hCAR]off-[hM4Di-mCherry]on-W3SL backbone construct containing the W3SL cassette (Addgene plasmid #111397).

Techniques: Injection, Construct, Over Expression, Virus, Expressing, RNAscope, Two Tailed Test

Journal: Nature Communications

Article Title: Stabilization of V1 interneuron-motor neuron connectivity ameliorates motor phenotype in a mouse model of ALS

doi: 10.1038/s41467-024-48925-7

Figure Lengend Snippet: a Cartoon depicting systemic administration via retroorbital intravenous injections of the viral constructs. b AAV vectors forcing overexpression of ESYT1 , mCherry , or eGfp were injected in SOD1 G93A ;En1 cre , En1 cre , and SOD1 G93A mice respectively at a volume of 100 μl per mouse. c – h Microphotograph of spinal cord ( c – d ), midbrain ( e – f ), and cortex ( g – h ) coronal sections of En1 cre mice showing cre-dependent mCherry overexpression upon AAV-PHP.eB-hSYN-DIO-mCherry-W3SL administration. mCherry protein investigation shows successful viral targeting of the lumbar segments of the spinal cord and significantly higher transfection of the spinal cord compared to midbrain and cortex (Nissl in light blue, mCherry in magenta). Higher magnification images are shown in ( d , f , h ). i Quantification of mCherry positive neurons, larger dots represent the mean per mouse, while smaller dots represent the number of positive neurons per hemi section (Repeated measures ANOVA and Tukey’s post hoc: SC vs midbrain P = 0.001, SC vs cortex P = 0.0021, midbrain vs cortex P = 0.149, n = 8 per mouse and N = 6, two-tailed). j – k Microphotograph of the mCherry overexpression in the ventral horns of the lumbar spinal cord of an En1 cre mouse upon systemic administration of the viral vector, using RNAscope. mCherry expression is specific to En1 positive neurons, thus selective for V1 interneurons (DAPI in blue, mCherry in orange, En1 in red, Chat in green). l Schematic depiction of the open field test (OFT), mice were assessed for general locomotor activity with the aid of automated tracking. m OFT revealed no significant differences in the distance traveled in any of the experimental conditions suggesting no deleterious effect after systemic delivery (One-way ANOVA and Dunnett’s post hoc: WT P = 0.0841, En1 cre P = 0.1674, SOD1 G93A ;En1 cre P = 0.8586, WT N = 6, En1 cre N = 4, SOD1 G93A N = 6, SOD1 G93A ;En1 cre N = 4). Scale bar in c = 100 μm and in j = 200 μm. All graphs show mean values ± SEM. Source data are provided as a Source Data file.

Article Snippet: The AAV overexpressing ESYT1 was developed utilizing a pAAV-hSYN-DIO[hCAR]off-[hM4Di-mCherry]on-W3SL backbone construct containing the W3SL cassette (Addgene plasmid #111397).

Techniques: Construct, Over Expression, Injection, Transfection, Two Tailed Test, Plasmid Preparation, RNAscope, Expressing, Activity Assay