Fig. 3 |. ENS neurotransmission is disrupted by pathologic α-Syn and restored by GBA1 gene transfer.

a, Triple vector scheme to express jRGECO1a calcium indicator and Channelrhodopsin (ChR2-EYFP) in peripheral neurons via packaging in AAV-PHP.S. b, Immunohistochemical labeling depicts transduction efficiency of vector system to express jRGECO1a (αRFP) and ChR2-EYFP (αGFP). Open arrows mark co-expression and closed arrow marks ChR2-EYFP expression alone. Experimental images were obtained from 6 independent mice, with similar results obtained. Scale bar, 50 μm. c, Immunohistochemical labeling depicts transduction efficiency of jRGECO1a (αRFP) in enteric neurons (PGP9.5). Experimental images were obtained from 6 independent mice, with similar results obtained. Scale bar, 25 μm. d, Representative image of an excised duodenal segment prepared for calcium imaging. e, Representative calcium trace plotted as percent change in fluorescence compared to pre-stimulation baseline fluorescence. Green traces indicate jRGECO1a⁺/ChR2⁺ neurons. f, Representative images of jRGECO1a fluorescence in duodenal enteric neurons recorded in (e) depicting baseline (left) and peak (right) calcium activity after the photostimulation pulse. Green arrows indicate jRGECO1a⁺/ChR2⁺ neurons. Experimental images were obtained from 6 independent mice, with similar results obtained. Scale bar, 50 μm. g, Peak percent change in fluorescence for individual duodenal jRGECO1a⁺-only or jRGECO1a⁺/ChR2⁺ neurons after photostimulation pulse. h, Cumulative probability plots of peak calcium responses shown in (g). i, Quantification of average peak percent change in fluorescence and area under the curve after photostimulation pulse for jRGECO1a⁺-only or jRGECO1a⁺/ChR2⁺ duodenal neurons before and after inoculation. Boxplots represent median, interquartile range, and 1.5× the interquartile range (AUC jRGECO1a⁺/ChR2⁺: WT vs. PFF, 7 dpi *p = 0.0488, 60 dpi *p = 0.0214; all ****p < 0.0001). j, Quantification of average peak percent change in fluorescence and area under the curve after photostimulation pulse for jRGECO1a⁺-only or jRGECO1a⁺/ChR2⁺ duodenal neurons before and after systemic delivery of AAV-PHP.S::ihSyn:GBA1. Boxplots represent median, interquartile range, and 1.5× the interquartile range (Peak ΔF/F jRGECO1a⁺: ASO, 0 vs. 60 dpvi ***p = 0.0007, 7 vs. 60 dpvi **p = 0.0068). P values were determined by one-way ANOVA (i) or two-way ANOVA (j). The following n values represents number of independent animals used for statistical evaluation: 3i, WT no stim = 2, WT stim = 3, PFF 7 dpi = 4, PFF 60 dpi = 3, monomer 7 dpi = 3, monomer 60 dpi = 3; 3j, all conditions = 3.