1. Volume 27, Issue 4, Pages 495-507 (February 2017)
Fast cAMP Modulation of Neurotransmission via Neuropeptide Signals and Vesicle Loading 
Wagner Steuer Costa, Szi-chieh Yu, Jana F. Liewald, Alexander Gottschalk 
Current Biology 
Volume 27, Issue 4, Pages (February 2017)
DOI: /j.cub
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2. Current Biology 2017 27, 495-507DOI: (10.1016/j.cub.2016.12.055)
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3. Figure 1
Photoactivating Cholinergic Neurons by bPAC Caused Exaggerated Locomotion and Required Intrinsic Depolarization
(A) bPAC::YFP expression in cholinergic neurons (punc-17 promoter), cell bodies and processes (arrows), and commissures (arrowheads).
(B) Mean (±SEM) crawling speed of bPAC-transgenic (black) and WT (gray) animals, before, during, and after photostimulation (blue bar).
(C) (Left, middle) Analysis of body bending, defined by three-point angles along the animal’s spine (13 points; 11 angles). (Right) Mean (±SEM) bending angles of animals in (B), shown as deviation from 180°, before, during, and after photostimulation.
(D) Body length analysis. 25 s bPAC activation evoked contraction (means ± SEM).
(E) Independent optogenetic photoactivation (via bPAC/blue light) or hyperpolarization (via the rhodopsin H+-pump MAC/yellow light).
(F and G) bPAC effects require depolarization. Coexpression of bPAC and MAC (dark blue and gray) or MAC only (black). Presence of the MAC chromophore all-trans retinal (ATR) is indicated. Analysis (mean ± SEM) of body length (F) and locomotion speed (G) before and during 25 s yellow/blue illumination (color bars) is shown.
In all panels, statistically significant differences are indicated (∗p ≤ 0.05, ∗∗p ≤ 0.01, and ∗∗∗p ≤ 0.001). (B and C): Student’s t test/Bonferroni correction is shown; (D, F, and G): two-way ANOVA/Bonferroni correction. See also Figure S1 and Movie S1.
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4. Figure 2
bPAC Stimulation Promoted SV and DCV Depletion as well as Endocytic LV Generation, whereas ChR2 Stimulation did Not Evoke DCV Release
(A) Representative 40-nm-thin section electron micrographs of cholinergic NMJs expressing bPAC (no treatment or 5, 30, and 300 s photostimulation, followed by 4 s until HPF fixation). Dense projection (DP) is shown; docked SVs, dense core vesicles (DCVs), and endocytic large vesicles (LVs) are indicated by black arrowheads and white and black arrows, respectively.
(B) Serial 3D reconstructions of non-stimulated and 30 s stimulated bPAC synapses, with synaptic structures as indicated.
(C) Synaptic structures as in (B) were outlined in each image, enabling analysis of numbers and spatial parameters.
(D–K) Statistical analysis of synaptic structures observed per profile (normalized to synapse area or perimeter in D and H) in bPAC- (D–G) and ChR2-expressing synapses (H–K; two types of ChR2 stimulation; 9 [1+8] s using ChR2(C128S) or 30 s using ChR2(H134R)): docked SVs (D and H); total SVs (E and I); LVs (F and J); and DCVs (G and K). Means ± SEM; (D–G): ∗p ≤ 0.05, ∗∗p ≤ 0.01, and ∗∗∗p ≤ 0.001; one-way ANOVA/Tukey correction; (H–K) unpaired t test.
See also Figure S2.
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5. Figure 3
bPAC Stimulation Induced Docked SV Depletion and SV Mobilization
(A and B) The distance along the PM from docked SVs to the closest DP (as illustrated in A) was summed in 33 nm bins, comparing unstimulated to 5, 30, and 300 s bPAC-stimulated synapses (B).
(C and D) The radial distance of cytosolic SVs (excluding docked SVs) to the closest DP, as illustrated in (C), was binned and summarized (D), as in (B).
(E) Empirical cumulative distribution function (eCDF), plotted from data in (D), showing for each profile the summed distance of all SVs to a DP, divided by the profiles’ area.
Mean numbers ± SEM per profile in (B) and (D), normalized to synapse perimeter (B) or area (D). (B and D): two-way ANOVA/Bonferroni correction or Kolmogorov-Smirnov test (E); ∗p ≤ 0.05, ∗∗p ≤ 0.01, and ∗∗∗p ≤ See also Figure S2.
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6. Figure 4 bPAC, but Not ChR2, Stimulation Induces DCV Release
(A) Neuropeptide secretion assay (left, scheme; right, fluorescence micrograph). NLP-21::Venus neuropeptide precursors are processed and released from cholinergic neurons and endocytosed by coelomocytes.
(B) Representative images of coelomocytes in control and bPAC animals, without and with photostimulation.
(C) Mean ± SEM coelomocyte fluorescence in WT, bPAC, and ChR2(C128S)-expressing animals, in dark or photostimulated, normalized to dark condition. One-way ANOVA/Bonferroni correction, ∗∗∗p ≤
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7. Figure 5 bPAC-Induced DCV Release Requires UNC-31/CAPS
(A) Coelomocyte fluorescence (mean ± SEM) assay as in Figure 4. unc-31(n1304) animals lacking CAPS cannot release neuropeptides after bPAC stimulation.
(B) Electron micrographs of WT and unc-31 mutant synapses expressing bPAC. DP, dense projection; black arrowheads, docked SVs; white arrows, DCVs.
(C) (Left) unc-31 synapses contain more DCVs and cannot release them in response to bPAC stimulation. (Right) Mean ± SEM. DCV numbers in 40 nm sections containing the DP, or flanking it, up to 800 nm axial direction are shown.
(D) unc-31 synapses contain less SVs, which are released after bPAC stimulation.
(E) Fluorescence of NLP-21::Venus in cell bodies (top) and nerve cord processes (bottom) in cholinergic neurons of the indicated genotype; representative micrographs.
(F) Quantification of fluorescence (mean ± SEM) of structures as shown in (E).
(G) Quantification of nerve cord fluorescence (mean ± SEM) in animals expressing bPAC and NLP-21::YFP in WT or unc-31(n1304), before and after 15 min photostimulation.
Student’s t test (F) or ANOVA (A, C, D, and G); Tukey-corrected; ∗p ≤ 0.05, ∗∗p ≤ 0.01, and ∗∗∗p ≤
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8. Figure 6
bPAC Stimulation Increased mPSC Rate and Amplitude, Requiring DCV Release, whereas ChR2 Increased Only mPSC Rate
(A and B) Original traces recorded by whole-cell patch-clamp in body-wall muscle cells during photoactivation (blue bar) of bPAC (A; increased mPSCs) or ChR2 (B; photo-EPSC followed by increased mPSC rate), expressed in cholinergic neurons. Gray, blue, and green tick marks indicate enlarged regions in color-boxed insets.
(C and E) Mean ± SEM mPSC rate (C) and amplitudes (E) in 1,000 ms bins, normalized to before illumination, for bPAC- (blue) and ChR2- (gray) expressing neurons. Illumination period is indicated by blue and gray bars for bPAC- and ChR2-stimulated neurons, respectively.
(D and F) Mean ± SEM mPSC rate (D) and amplitudes (F) before, during, and after photoactivation of animals expressing bPAC in WT (blue), unc-47, or unc-31 mutants (white or brown) or ChR2 in WT (gray), as well as non-transgenic WT controls (black). Also shown are acy-1(ce2) gain-of-function (g.o.f.) mutants (green) compared to WT (no transgene or stimulation). n = 7–11 animals.
(G and H) Cumulative distribution of mPSC amplitudes (n = 8) in bPAC- (G) or ChR2- (H) expressing animals, before (black), during (orange), or after the light stimulus (gray).
(I and J) As in (C) and (E), comparing unc-31 mutants to WT. Blue bar indicates illumination period.
ANOVA/Bonferroni correction; ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < See also Figures S3 and S4.
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9. Figure 7
bPAC-Stimulated Neuropeptide Release Acts Presynaptically to Increase SV Loading
(A and B) Potential mechanisms of bPAC-stimulated neuropeptide signaling to increase mPSC amplitudes, acting on (A) postsynaptic AChRs or (B) presynaptic SV ACh loading. Single ACh applications are shown, unless indicated (ACh rep., ACh was applied repetitively).
(C and D) Puff application of ACh (500 μM) on whole-cell patch-clamped body-wall muscle cells (in animals expressing bPAC in cholinergic neurons) induced currents (C, original trace; D, mean ± SEM), without or with preceding or concomitant bPAC photostimulation, in WT or unc-47 (vGAT) or unc-17 (vAChT) mutants, as indicated.
(E) Puff application of L-AChR-specific agonist levamisole, as in (D).
(F) Current decay analysis, ACh- or Lev-induced PSCs, and ratio of current at 1 s and peak current (mean ± SEM), without and with bPAC stimulation.
(G) Thousands of mPSCs, before, during, or after bPAC stimulation, were isolated from traces of WT (n = 7) or unc-31 animals (n = 9), aligned, and averaged (left; see Figure S5 for individual mPSCs and τon analysis). τoff was determined for each mPSC and averaged (mean ± SEM; right).
(H) Sequence of experiments with drug application analyzed in (I) and (J).
(I and J) Means ± SEM of mPSC rate and amplitude in animals expressing bPAC in cholinergic cells, with incubation of vehicle or bafilomycin A1 (BafA1; I) vATPase blocker or vAChT blocker vesamicol (Ves.; J), before or during photostimulation. See also Figure S6.
(K) SV diameter was measured for all SVs (n = 788–4,334; Figure S7A) and then averaged per profile (with at least ten SVs) as mean SV diameter (medians, interquartile range; whiskers: 2.5–97.5 percentile) for indicated genotypes and conditions of bPAC- or ChR2(C128S)-stimulated synapses.
(L) SV diameter in unc-11(e47) mutants, as in (K).
(M) Mean ± SEM mPSC rate and amplitudes compared in WT (black) and unc-11(e47) (yellow).
One-way ANOVA/Bonferroni correction (D) or Tukey correction (K); Student’s t test (E, F, and M); paired samples t test (I and J): ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001.
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