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. 2024 Jun 7;15:4867. doi: 10.1038/s41467-024-48925-7

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© The Author(s) 2024

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 7 — 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.