Fig 2: SgII puncta number and intensity are normal in the absence of Cgs. (a) Example images of control and CgA/B-/- hippocampal neurons (DIV16) stained for the dense-core vesicle marker SgII (green), the dendritic marker microtubule-associated protein 2 (MAP2) (blue) and the axonal marker SMI312 (red). SMI-positive (red) nuclei are the astrocytes nuclei, known to contain SMI immunoreactivity (Weigum et al. 2003). (b) Zoomed areas in the boxes shown in (a). (c) Quantification of the number of SgII puncta per neuron. (d) Quantification of the number of SgII puncta per µm per neurite. (e) Mean intensities of the SgII puncta per neuron. (f) Analysis of the total neurite length (MAP2 and SMI312 staining). (g) Sholl analysis showing the mean number of neurite branches against the distance from soma. (h) Analysis of the mean number of SgII puncta against the distance from soma. (Data shown as mean ± SEM. Control n = 12 neurons and CgA/B-/- n = 12 neurons. (c and f) Student's t-test, (d and e) MW-U test.

Fig 3: The size and number of synaptic dense-core vesicles (DCVs) is normal in the absence of Cgs. (a) Example electron micrographs of control and CgA/B-/- hippocampal neurons (DIV19). DCVs are indicated with arrows (b) Quantification of the mean diameter of DCVs. (c) Percentage distribution of the number of DCVs found per synapse. (Data shown as mean ± SEM. For size measurements: Control n = 9 neurons (660 DCVs) and CgA/B-/- n = 9 neurons (521 DCVs); for DCV distribution in synapses, Control n = 9 neurons (377 synapses) and CgA/B-/- n = 9 neurons (399 synapses, from three independent cultures). Student's t-test.

Fig 4: Dense-core vesicle (DCV) fusion events are less frequent, but fusion probability is normal in the absence of Cgs. (a) Example of a neuropeptide Y (NPY)-pHluorin release event (arrowhead) in a control neurite and the unquenching of all the DCVs present in the area upon NH 4Cl puff (arrows). Scale bar: 2 µm. (b) Averaged frequency distribution of the events against the time of recording. SEM plotted as dotted line. The stimulation is shown in gray (16 bursts of 50 action potentials at 50 Hz). (c) Averaged cumulated fusion events per neuron. (d) Normalized cumulated fusion events. (e) Quantification of the total number of fusion events per neuron. (f) Total DCV pool per neuron calculated from NH 4Cl puff signal. (g) Fusion probability of DCVs of both genotypes. (h) NPY-pHluorin puncta intensity in the NH 4Cl puff. (Data are shown as mean ± SEM. MW-U test *p = 0.0477. Control n = 29 neurons and CgA/B-/- n = 36 neurons from four independent cultures. (e, g, and h) MW-U test. (f) Student's t-test.

Fig 5: Duration and amplitude of fusion events is normal in the absence of Cgs. (a) Sequential frames showing examples of two types of neuropeptide Y (NPY)-pHluorin release events in control neurons. Scale bar: 1 µm. Quantification of each event is shown as ?F/F0. (b) Like in (a) but for CgA/B-/- neurons. (c) Averaged histograms of the duration of the NPY-pHluorin events. Inset: Same histograms plotted separately. (d) Mean duration of the events per cell. (e) Percentage of transient versus persistent events in both genotypes. (f) Classification of the events depending on the duration: short transient (=1 s), long transient (> 1 s to = 5 s), short persistent (> 5 s to = 10 s), and long persistent (> 10 s). (g) Averaged histograms of the amplitude of the NPY-pHluorin events. Inset: Same histograms plotted separately. (h) Quantification of the mean amplitude of the events per neuron. Data are shown as mean ± SEM. Control n = 29 neurons (3321 events) and CgA/B-/- n = 36 neurons (2480 events) from four independent cultures. (d and h) MW-U test. (e) Student's t-test.