1. Volume 92, Issue 6, Pages 1204-1212 (December 2016)
Acute Fasting Regulates Retrograde Synaptic Enhancement through a 4E-BP- Dependent Mechanism 
Grant Kauwe, Kazuya Tsurudome, Jay Penney, Megumi Mori, Lindsay Gray, Mario R. Calderon, Fatima Elazouzzi, Nicole Chicoine, Nahum Sonenberg, A. Pejmun Haghighi 
Neuron 
Volume 92, Issue 6, Pages (December 2016)
DOI: /j.neuron
Copyright © 2016 Elsevier Inc. Terms and Conditions

2. Figure 1 Loss of 4E-BP Enhances Baseline Synaptic Transmission
(A) Representative traces of mEJCs and EJCs from control (Thor1Rev1), 4E-BPΔ (Thor2), 4E-BP1 (Thor1), and 4E-BP/Df (Thor2/Df(2L)tim-02) larvae.
(B) Quantification of mEJC, EJC, and QC from genotypes described in (A) and heterozygous genotype 4E-BPΔ/+ (Thor2/+). n = 20, 11, 20, 17, 20. One-way ANOVA.
(C) Representative traces of EJCs from wild-type (w1118) and 4E-BPΔ mutant (Thor2;MHC/+) larvae, as well as 4E-BPΔ mutant larvae rescued by transgenic overexpression of 4E-BP in the muscle (Thor2;MHC-Gal4/UAS-4E-BP) or in motor neurons (BG380-Gal4/+; Thor2; +/UAS-4E-BP).
(D) Quantification of mEJC, EJC, and QC from genotypes described in (C). n = 20, 21, 21, 20. One-way ANOVA.
(E) QC measured by failure analysis in control (Thor1Rev1) and 4E-BPΔ (Thor2) larvae. n = 11, 12. Student’s t test.
(F) Representative images of antibody staining against Brp and Hrp at muscles 6/7 in control (Thor1Rev1) and 4E-BPΔ (Thor2) larvae. Scale bar represents 10 μm.
(G) Quantification of the total number of type 1 synaptic boutons per muscle surface area (n = 19, 14; p = 0.257) and active zone density per synaptic area (n = 10, 11; p = 0.216) at muscles 6/7 in control (Thor1Rev1) and 4E-BPΔ (Thor2).
Error bars represent SEM. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < See Table S1.
Neuron  , DOI: ( /j.neuron )
Copyright © 2016 Elsevier Inc. Terms and Conditions

3. Figure 2
Acute Fasting Enhances Transcription of 4E-BP to Suppress Retrograde Synaptic Compensation
(A) Representative traces of mEJCs and EJCs from wild-type (w1118) larvae from food (6 hr) or no food conditions (6 hr) and GluRIIA−/− mutant larvae (GluRIIASP16/Df(2L)Cl−h4) from food (6 hr) or no food (6 hr) conditions.
(B) Quantification of mEJC, EJC, and QC from the genotypes and conditions shown in (A). n = 18, 19, 17, 20. One-way ANOVA.
(C) Quantification of mEJC, EJC, and QC from GluRIIA−/− mutant larvae (GluRIIASP16/Df(2L)Cl−h4) with 0, 0.5–1, 3–4, and 6–7 hr of no food. n = 14, 13, 13, 17. One-way ANOVA.
(D) Representative images of antibody staining against Brp and Hrp at muscles 6/7 in GluRIIA−/− mutant larvae (GluRIIASP16/Df(2L)Cl−h4) from food (6 hr) or no food (6 hr) conditions. Scale bar represents 10 μm.
(E) Quantification of the total number of type 1 synaptic boutons per muscle surface area (n = 22, 19; p = 0.743) and active zone density per synaptic area (n = 10, 13; p = 0.481) at muscles 6/7 in GluRIIA−/− mutant larvae (GluRIIASP16/Df(2L)Cl−h4) following 6 hr of fasting (no food), normalized to control (GluRIIA mutant larvae 6 hr transfer on standard food [food]).
(F) Relative mRNA expression levels of 4E-BP in muscle tissue from wild-type (w1118) larvae from food (6 hr) or no food (6 hr) conditions. N (experiments) = 6. Student’s t test.
(G) Muscle 6 stained with anti-βGal (green) in Thor-LacZ larvae (Thor-LacZ/+;MHC-Gal4/+) from food (6 hr) or no food (6 hr) conditions. Scale bar represents 20 μm.
(H) Quantification of nuclear LacZ intensity from the conditions shown in (G), normalized to food condition. N (experiments) = 3, 3; n > 200 nuclei.
(I) Example western blots comparing the levels of non-phosphorylated 4E-BP and phosphorylated 4E-BP in muscle tissue from wild-type (w1118) larvae treated in food (6 hr) or no food (6 hr) conditions.
(J) Quantification of the proteins levels of non-phosphorylated 4E-BP with respect to actin in wild-type (w1118) larvae under food (6 hr) or no food (6 hr) conditions. N (experiments) = 4. Student’s t test.
(K) Representative traces of mEJCs and EJCs from GluRIIA−/− mutants heterozygous for 4E-BP (GluRIIASP16, Thor1/Df(2L)Cl−h4) under food (6 hr) or no food (6 hr) conditions.
(L and M) Quantification of mEJC, EJC, and QC from the genotype and conditions shown in (K; n = 19, 20) and from 4E-BP−/− mutant larvae (Thor1) kept under food (6 hr) or no food (6 hr) conditions. n = 8, 13. Student’s t test.
Error bars represent SEM. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < See Table S1.
Neuron  , DOI: ( /j.neuron )
Copyright © 2016 Elsevier Inc. Terms and Conditions

4. Figure 3
Postsynaptic Foxo Is Required for the Increase in 4E-BP Transcription Triggered by Acute Fasting
(A) Muscle 6 stained with anti-βGal (green) from food (6 hr) or no food (6 hr) conditions in Thor-LacZ larvae expressing RNAi against Foxo in the muscle (Thor-LacZ/+; MHC-Gal4/UAS-FoxoRNAi). Scale bar represents 20 μm.
(B) Quantification of nuclear LacZ intensity from the conditions shown in (A), normalized to food condition. N (experiments) = 5, 5; n > 250 nuclei.
(C–F) Representative traces of mEJCs and EJCs from control larvae expressing Foxo RNAi in the muscle (C, MHC-Gal4/UAS-FoxoRNAi), GluRIIA−/− mutant larvae expressing Foxo RNAi in the muscle (D, GluRIIASP16/Df(2L)Cl−h4; MHC-Gal4/UAS-FoxoRNAi) or fasted for 6 hr (E), and GluRIIA−/− mutant larvae expressing Foxo RNAi in the muscle with heterozygosity for eIF4E (F, GluRIIASP16/Df(2L)Cl−h4; MHC-Gal4, eIF4Es058911/UAS-FoxoRNAi) fasted for 6 hr.
(G) Quantification of mEJC, EJC, and QC from the genotypes and conditions shown in (C), (D), (E), and (F). n = 25, 15, 18, 16. One-way ANOVA.
Error bars represent SEM. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < See Table S1.
Neuron  , DOI: ( /j.neuron )
Copyright © 2016 Elsevier Inc. Terms and Conditions

5. Figure 4
Enhanced Synaptic Transmission in 4E-BP Mutants Requires eIF4E
(A) Muscle 4 NMJs stained with anti-GFP (white) and anti-Dlg (green) in control (Thor1Rev1;eIF4E::GFP /+) and 4E-BPΔ (Thor2;eIF4E::GFP/+) larvae. Scale bar represents 10 μm.
(B) Quantification of fluorescent intensity of GFP with respect to Dlg from the genotypes shown in (A), normalized to control. Student’s t test.
(C) Representative traces of mEJCs and EJCs from 4E-BPΔ mutant larvae (Thor2), 4E-BP−/− larvae heterozygous for eIF4E (Thor2/ Df(2L)tim-02; eIF4Es058911/+), and 4E-BPΔ larvae heterozygous for S6K (Thor2;S6Kl-1/+).
(D) Quantification of mEJC, EJC, and QC from genotypes described in (C). n = 20, 12, 8. One-way ANOVA.
(E) Quantification of mEJC, EJC, and QC from 4E-BPΔ mutant larvae (n = 21) expressing only MHC-Gal4 (Thor2;MHC-Gal4/+), expressing 4E-BP transgene in muscle (Thor2;MHC-Gal4/UAS-4E-BP) (n = 20), or expressing 4E-BPaa transgene in muscle (Thor2/Thor1,UAS-4E-BPaa;MHC-Gal4/+) (n = 12). Student’s t test.
Error bars represent SEM. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < See Table S1.
Neuron  , DOI: ( /j.neuron )
Copyright © 2016 Elsevier Inc. Terms and Conditions