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. 2020 Sep 9;11:4510. doi: 10.1038/s41467-020-18140-1

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© The Author(s) 2020

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 license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license 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 license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 4 — a Representative whole-mount images of the pre- (neurofilament/synaptophysin or YFP) and postsynapse (acetylcholine receptors (AChRs) stained using alpha bungarotoxin) in extensor digitorum longus (EDL) muscle for 10-month-old control (10mCON), 30-month-old (30mCON), and 30-month-old mice treated from 15 months with rapamycin (30mRM, left) and for 9-month-old control (9mCON), TSCmKO (9mTSCmKO), and TSCmKO + 4 weeks of rapamycin-treatment (TSCmKO+RM, right) mice. Morphological properties of whole-mount EDL NMJs, including b axon diameter, c the percentage of NMJs with sprouting axons, and d the number of postsynaptic AChR clusters for 9mTSCmKO and sarcopenic experiments. e Acetylcholine receptor density in EDL cross sections. f–h Morphological properties of whole-mount GAS NMJs for 10mCON, 30mCON, and 30mRM mice. In situ electrophysiological readouts of individual NMJ transmission properties in the tibialis anterior (TA) and soleus (SOL) muscle of 11-month-old TSCmKO mice and littermate controls, including (i) miniature end- plate current (mEPC), j end-plate current (EPC), k quantal content, and l EPC over repeated stimulations. m EMG recordings of compound muscle action potential (CMAP) rundown over four consecutive stimulations in the GAS of 9-month-old TSCmKO mice and littermate controls at stimulation frequencies between 3 and 15 Hz. Nerve- relative to muscle-stimulated peak tetanic force in SOL nerve-muscle preparations in (n) 10- (n = 5) and 28–30-month-old (n = 13) wild-type mice, and (o) 9-month-old control (n = 11), TSCmKO (n = 10), and TSCmKO mice treated with rapamycin for 4 weeks (n = 5). Group numbers for 10mCON are n = 5 (b, d), 3 (c), 6 (e), and 4 (f–h) mice. For 30mCON and 30mRM, n = 5 (b, d), 4 (c, f–h), and 7 (e) mice. For 9mCON, 9mTSCmKO, and 9mTSCmKO+RM, n = 4 (b–d) and 3 (e) mice. For 11mCON and 11mTSCmKO, n = 5 (TA: i–l) and 4 (SOL: i–l) mice. For m, n = 6 (12mCON) and 5 (12mTSCmKO) mice. For n, n = 5 (10m) and 13 (28–30 m). For o, n = 11 (9mCON), 10 (9mTSCmKO), and 5 (9mTSCmKO + RM) mice. Data are presented as mean ± SEM. One-way ANOVA with Fisher’s LSD post hoc test (b–h, o), two-way (i–j) or two-way repeated-measure ANOVA (l, m) with Fisher’s LSD post hoc test (m), or two-tailed independent student’s t test (n) were used to compare between data. *, **, and *** denote a significant difference between groups of P < 0.05, P < 0.01, and P < 0.001, respectively. Where 0.05 < P < 0.1, P values are reported.