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. 2023 Jul 24;14:4452. doi: 10.1038/s41467-023-40142-y

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© The Author(s) 2023

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. 8 — a Schematic of heat-shock induced Is MN ablation protocol. b Cartoon representation of a dissected larva (pink) and a hemisegment highlighted by dashed red rectangle. The target muscle examined in this figure is shown in blue. Cartoon is generated with Biorender. NMJs of MN4-Ib in late third instar larvae (hs-FLP,UAS-GFP/ + ;UAS-FRT stop FRT-hid-2A-rpr/+;Is-GAL4/+) with (c) no heat-shock, (d) embryo heat-shock, (e) first instar heat-shock, (f) second instar heat-shock and (g) third instar heat-shock, stained with GFP (green), HRP (magenta), and DLG (gray). In embryos and first and second instar heat-shocked larvae, the Is NMJs were fully cleared, while Is synaptic debris remains in third instar heat-shocked larvae. Arrows indicate the Is MN (c) or Is MN debris (g). h Quantification of MN4-Ib bouton number in late third instar larvae with Is MNs ablated at different developmental stages. F(4, 127) = 10.23, p < 0.0001, One-way ANOVA. Control vs embryo heat-shock, p < 0.0001. Control vs first instar heat shock, p = 0.3021. Control vs second instar heat-shock, p = 0.7310. Control vs third instar heat-shock, p = 0.8756. N (NMJs) = 33, 36, 13, 25, 25. i Comparison of the normalized EPSP of late third instar larvae with Is MNs ablated at different developmental stages to Ib/Ib+Is baseline. Embryo heat-shock, t(27) = 7.126, p < 0.0001, unpaired t test, two-tailed. First instar heat-shock, t(21) = 6.622, p < 0.0001, unpaired t test, two-tailed. Second instar heat-shock, t(22) = 9.485, p < 0.0001, unpaired t test, two-tailed. Third instar heat-shock, t(23) = 8.557, p < 0.0001, unpaired t test, two-tailed. j Quantification of normalized quantal content of late third instar larvae with Is ablated at different developmental stages. F(4, 67) = 9.109, p < 0.0001, One-way ANOVA. Embryo heat-shock vs third instar heat-shock, p = 0.0002. Non-significant for the others. This result suggested an increase of cross-neuron plasticity when acutely ablated Is MNs. For (i, j), N (NMJs) = 19, 17, 11, 12, 13. Error bars indicate ± SEM, ns = non-significant, ***p < 0.001, ****p < 0.0001.