Pool size estimations for dense-core vesicles in mammalian CNS neurons (2018)
Figure 3

Figure 3 - DCVs are present in axonal, dendritic and synaptic compartments.

A-C Representative confocal images and zooms of a single isolated hippocampal neuron (DIV14) immunostained for the endogenous DCV marker ChgB (green), dendritic marker MAP2 (red) and ß3-tubulin (blue) as neurite marker. Areas of zooms are indicated in (A). Closed arrows: dendritic ChgB staining, open arrows: axonal ChgB staining. Scale bars: 20 µm (A), 5 µm (B, C).

D Zoom of immunostaining for the excitatory synaptic vesicle protein VGLUT1 and DCV marker ChgB. Closed arrows: synaptic ChgB staining, open arrows: extra-synaptic ChgB staining. Scale bar: 5 µm.

E Zoom of immunostaining for two synaptic proteins VGLUT1 and Synapsin I/II. Scale bar: 5 µm.

F Manders' coefficients of colocalization analysis between ChgB, ß3-tubulin and MAP2.

G Manders' coefficients of colocalization analysis between ChgB and VGLUT1, with VGLUT1-Synapsin I/II colocalization as positive control and VGLUT1-Synapsin I/II rotated 90° as negative control.

H-M Distribution of DCVs in single hippocampal neurons (DIV14) stained for ChgB, MAP2 and ß3-tubulin. Dendrites were determined as MAP2 positive neurites and axonal measurements were calculated by subtracting the dendritic population from the total pool measured in β3-tubulin positive neurites. (H, I) Number of ChgB puncta per neuron in dendrites, axons and the total pool (dendrites + axons). Wilcoxon matched-pairs signed rank test: n.s. = not significant, p = 0.75. (J) Total dendritic and axonal length (mm) per neuron. Wilcoxon matched-pairs signed rank test: **** p = < 0.0001. (K) ChgB puncta per μm dendrite and axon per neuron. Lines next to individual data points show mean ± SEM. Wilcoxon matched-pairs signed rank test: **** p = < 0.0001. (L) Mean ChgB fluorescence intensity per puncta in the total neuron (dendrites + axons) and in dendrites only. Wilcoxon matched-pairs signed rank test: **** p = < 0.0001. (M) Histogram of fluorescence intensity of ChgB puncta in the total neuron (dendrites + axons) and in dendrites only.

N-O Distribution of DCVs in single hippocampal neurons (DIV14) stained for ChgB, MAP2 and the axonal marker SMI312. ChgB puncta were measured in dendrites, determined as MAP2 positive neurites, and in axons, determined as SMI312 positive neurites. (N) ChgB puncta per μm dendrite and axon per neuron. Lines next to individual data points show mean ± SEM. Wilcoxon matched-pairs signed rank test: **** p = < 0.0001. (O) Mean ChgB fluorescence intensity per puncta in dendrites and axons. Wilcoxon matched-pairs signed rank test: **** p = < 0.0001.

P-Q Distribution of DCVs in dendrites and axons of sparse-labeled network cultures. (P) ChgB puncta per μm dendrite or axon. Lines next to individual data points show mean ± SEM. Mann-Whitney U test: **** p = < 0.0001. (Q) Mean ChgB fluorescence intensity per puncta in dendrites and axons. Unpaired t-test: *** p = < 0.0001.

R-S Analysis of DCVs in axons and dendrites of mCherry labeled mouse Dentate Gyrus (DG) granule cell tissue after stereotaxic viral injections and post-hoc immunostained for ChgA as DCV marker. (R) Representative mouse brain slice with labeled DG granule cells (mCherry-filler, red) and immunostained for ChgA (green). ChgA puncta were measured in dendrites and axons at indicated areas. Scale bar: 250 µm. (S) ChgA puncta per μm dendrite or axon in DG granule cells. N = 1 mouse (6 dendrite regions, 7 axonal regions).

Data information: Bars show mean ± SEM, dots represent measurements of individual single neurons or neuronal regions. N numbers represent number of experiments and number of (single) neuron observations are in brackets.