Figure 2.

PRRT2 Knockdown Decreases Synapse Density and Increases Docked SVs in Low-Density Hippocampal Neurons

(A) Representative images of dendrites of hippocampal neurons infected at 7 DIVs with Scr, Sh4, and Sh4 + Sh4-resistant PRRT2 (Sh4+PRRT2) or left uninfected and analyzed at 14 DIVs. Synaptic boutons were identified by double immunostaining for Bassoon (Bsn, red) and Homer1 (green). The colocalization panels (Col. points) highlight the double-positive puncta (black) corresponding to synapses. Scale bar, 10 μm.

(B) Quantitative analysis of synaptic puncta counted on 30-μm dendrite tracts starting from the cell body in neurons treated as in (A). Data are means ± SEM from three independent experiments, each carried out in duplicate. Five dendrites for each neuron, from at least ten neurons for each sample, were counted. ^∗p < 0.05, one-way ANOVA/Bonferroni’s multiple comparison test. NI, not infected.

(C). Conventional TEM analysis of nerve terminals from PRRT2 KD hippocampal neurons revealed an increase in docked SVs and a preservation of the total SVs with respect to control neurons. Shown are representative TEM images of nerve terminals from neurons transduced with either Scr or Sh4 at 7 DIVs and fixed/processed at 14 DIVs. Scale bar, 200 μm.

(D) Quantitative TEM analysis of the synaptic density from serial ultrathin sections. The volume density of symmetric and asymmetric synapses was calculated from the 2D count of synaptic profiles in sections from Sh4- (red bars) and Scr-treated (black bars) neurons and is expressed as mean (± SEM) number of synapses per square micrometer.

(E). 3D reconstructions of synaptic terminals from serial ultrathin sections confirm the increase in the number of docked SVs in low-density neuronal cultures. Shown are representative 3D reconstructions from 60-nm-thick serial sections obtained from Scr-treated (left) and PRRT2 KD (right) synapses. Total SVs and SVs physically docked at the AZ are depicted as blue and red spheres, respectively. The AZ and mitochondria are shown in yellow and green, respectively. Scale bar, 200 nm.

(F and G) Morphometric analysis of three-dimensionally reconstructed synapses. PRRT2 KD synapses (red bars) displayed an increased number of AZ-docked SVs (F) and a preserved total number of SVs (G) with respect to Scr-treated synapses (black bars).

(H) Spatial distribution of SVs in nerve terminals of Scr-treated (black symbols) and Sh4-treated (red symbols) neurons. The mean (± SEM) number of SVs located within successive 50-nm shells from the AZ and normalized by the total SV content of each terminal is given as mean ± SEM as a function of the distance from the AZ.

(I) Morphometric analysis of SV diameter. PRRT2 KD synapses (red bars) displayed a smaller SV size with respect to Scr-treated synapses (black bars).

Nerve terminal areas (0.689 ± 0.044 μm² and 0.741 ± 0.035 μm² for Scr- and PRRT2 sh-RNA-infected neurons, respectively) and AZ lengths (0.302 ± 0.023 μm and 0.347 ± 0.013 μm for Scr- and PRRT2 sh-RNA-infected neurons, respectively) were similar in the two experimental groups (140 and 150 synapses for Scr and PRRT2 shRNA-infected neurons, respectively, from three independent preparations). ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001, unpaired Student’s t test (E, G, and H) and Kolmogorov-Smirnov test (F). See also Figure S2.