Panel A shows a schematic illustrating synaptic vesicle reserve pools surrounding the photoreceptor ribbon synapse. Panel A prime shows a schematic illustrating synaptic vesicle distance measurements relative to the synaptic ribbon. Panel A double prime shows a schematic illustrating synaptic vesicle density measurements along the synaptic ribbon. Panel A triple prime shows a schematic illustrating cytoplasmic reserve pool synaptic vesicle density measurement region. Panel B shows transmission electron microscopy images of wild type ribbon reserve pool with enlarged synaptic ribbon view. Panel C shows transmission electron microscopy images of knockout ribbon reserve pool highlighting altered vesicle organization near ribbon. Panel D shows transmission electron microscopy images of wild type cytoplasmic reserve pool with enlarged vesicle distribution view. Panel E shows transmission electron microscopy images of knockout cytoplasmic reserve pool with enlarged vesicle distribution view. Panel F shows a bar graph comparing synaptic vesicle distance to ribbon between wild type and knockout. Panel G shows a histogram comparing synaptic vesicle distribution by distance from ribbon in both genotypes. Panel H shows a bar graph comparing synaptic vesicle density along ribbon between wild type and knockout. Panel I shows a bar graph comparing cytoplasmic reserve pool vesicle density between wild type and knockout. Panel J shows high pressure freezing electron microscopy images of wild type photoreceptor ribbon synapses at sixty kilovolt. Panel K shows high pressure freezing electron microscopy images of knockout photoreceptor ribbon synapses at sixty kilovolt. Panel L shows high pressure freezing electron tomography virtual section images of wild type ribbon synapse with enlarged inset. Panel L prime shows a schematic illustrating tethered and untethered synaptic vesicle organization around the ribbon. Panel M shows high pressure freezing electron tomography virtual section images of knockout ribbon synapse with enlarged inset. Panel M prime shows 3D model of electron tomograms illustrating tethered and untethered synaptic vesicle organization in knockout synapse. Panel N shows a stacked bar graph comparing proportions of tethered and untethered synaptic vesicles between genotypes.
Loss of Piccolino results in altered distance and density of SVs and ablates SV tethering at rod photoreceptor SRs. (A) Schematic of a rod photoreceptor ribbon synapse with SV pools: RRP (yellow), ribbon RP (blue), and cytoplasmic RP (gray). (A′–A‴) Schematic representation of methodology used for measuring SV distance (A′) and SV density in the ribbon RP (A″) and the cytoplasmic RP (A‴). Quantified parameters are indicated by dashed red lines. (B and C) Representative EM micrographs of PcloWT (B) and PclocKO (C) rod photoreceptor SRs. Red arrowheads (➤) indicate SVs in direct contact with the SR surface; a black arrowhead (➤) indicates a disrupted SV halo. (D and E) Representative EM micrographs of SVs in PcloWT (D) and PclocKO (E) rod photoreceptor terminals. (F and G) Quantification (F) and frequency distribution (G) of the distance between SVs in the ribbon RP and the SR in PcloWT and PclocKO rod photoreceptors. Data are mean ± SD, **P = 0.0014, unpaired t test, n = 66–78 terminals per animal, three animals. (H) Quantification of SV density in the ribbon RP (SVs per µm SR) in PcloWT and PclocKO rod photoreceptors. Data are mean ± SD, **P = 0.0050, unpaired t test, n = 66–78 terminals per animal, three animals. (I) Quantification of SV density in the cytoplasmic RP (SVs per 0.04 µm2) in PcloWT and PclocKO rod photoreceptors. Data are mean ± SD, n.s., P = 0.8579, unpaired t test, n = 62–74 terminals per animal, three animals. (J and K) Representative conventional TEM micrographs of HPF/FS-prepared PcloWT (J, i–iii) and PclocKO (K, i–iii) rod photoreceptor terminals, confirming recapitulation of the immersion-fixed phenotype: a halo of SVs at a distance from the SR in PcloWT and its absence in PclocKO. (L–M′) Representative EM tomogram virtual sections and 3D reconstructions of a PcloWT (L and L′) and a PclocKO rod photoreceptor SR (M and M′). (N) Quantification of SVs connected (+tether, blue) or not connected (−tether, orange) to SRs in PcloWT and PclocKO rod photoreceptors. Data are ± SD, ****P < 0.0001, unpaired t test, n = 10 tomograms for each genotype, two animals. Scale bar = 200 nm in C for B and C and in E for D and E, 100 nm in high power views and in L–M′. HC, horizontal cell; BC, bipolar cell.