PMC

Endocytosis of FM dye by AP180- and CALM KD-neurons. Live neurons expressing Syp::EGFP (green) and AP180shRNA (a; AP180 KD) or CALMshRNA (b; CALM KD) were incubated with fixable FM 4–64 (magenta) and then fixed. Although the Syp::EGFP puncta are considerably smaller in AP180- and CALM KD-neurons, these smaller puncta internalize the FM dye (white arrowheads). The experiment was repeated three times (Color figure online)

AP180- and CALM-knocked down (KD) neurons have smaller presynaptic puncta. Neurons were transfected on 16 div (days in vitro) and examined 4 days later. a left, confocal image of a hippocampal neuron expressing Syp::EGFP; right, the Syp::EGFP-positive puncta (green) contain the endogenous SV protein Synapsin1 (Syn1; magenta) and co-localize with endogenous active zone protein Bassoon (magenta). b Neurons were co-transfected with Syp::EGFP and empty shRNA vector (Control) or AP180shRNA (AP180 KD). Additional examples are shown in . Results using a different AP180shRNA are shown in . Cumulative frequency plot of puncta size and the average size (insert) shows smaller puncta in AP180 KD-neurons. c Synaptic puncta of CALM KD-neurons are also smaller. Additional examples are shown in , and results using a different CALMshRNA are shown in . For each experiment, at least 200 axonal segments (exemplified in b and c) from 8 to 10 coverslips obtained from 4 to 5 cultures were analyzed. Data represent mean ± SEM. ***p<0.001 (Color figure online)

Synaptic vesicles of CALM KD-neurons. Neurons were transfected with CALMshRNA together with EGFP–mHRP. Transfected neurons have dark DAB reaction products filling their presynaptic terminals (pre) in some cases (white letters in a and b), or the labeling is only on the presynaptic membranes in other cases (black arrowheads in c–g). de, dendrite; sp, spines. Nontransfected presynaptic terminals opposing nontransfected postsynaptic terminals from the same culture are used as the controls (h). Synaptic vesicles from the CALM KD-neurons are distinctly variable in size and shape. Scale bar in h is 100 nm and it applies to all micrographs. i Histogram shows broader size distribution of synaptic vesicles from the CALM KD-neurons. Total of 274 potential EM micrographs from two cultures were analyzed (control, n = 1,010 synaptic vesicles; CALM KD, n = 3,212 synaptic vesicles). j The average size of synaptic vesicles of the CALM KD-neurons is significantly increased. k shows reduced synaptic vesicle density in the CALM KD-neurons. l shows unchanged presynaptic terminal size. m shows significantly reduced SV cluster size. ***p<0.001; **p<0.01. Data represent mean ± SEM

Presynaptic terminals of cultured hippocampal neurons revealed by electron microscopy. Following co-transfection with empty shRNA vector and EGFP–mHRP, neurons were processed for DAB detection and then examined by electron microscopy. Some presynaptic terminals (pre) of transfected neurons are filled with dark DAB reaction products (denoted by white letters in a and b), while other presynaptic terminals have DAB reaction products only on the plasma membrane or a part of the membrane (denoted by black arrowheads in c and d). Ultrastructural characteristics are not visibly different between the DAB-filled or DAB-outlined presynaptic terminals. Densely packed synaptic vesicles are homogenous in size and shape. Endosomes (en) are seen in some presynaptic terminals. There is also no difference between the presynaptic terminals that oppose transfected (DAB labeled) or nontransfected (no DAB labeling) postsynaptic dendrites (de) or spines (sp). Synapses formed from nontransfected presynaptic terminals onto nontransfected post-synaptic terminals (from the same culture) serve as controls (e–g). Scale bar in g is 100 nm, and it applies to all micrographs. h and i show size distribution and average size of SVs. Data are pooled from DAB-filled and DAB-outlined presynaptic terminals. Total of 306 potential EM micrographs from two cultures were analyzed (control, n = 1,160 synaptic vesicles; vector, n = 2,511 synaptic vesicles). j The density of SVs was calculated by counting the number of synaptic vesicles in randomly selected areas and normalizing to per lm². k The size of presynaptic terminals. l The size of SV clusters. Data represent mean ± SEM

Synaptic vesicles of AP180 KD-neurons. (Notice that all micrographs presented in this figure are *20 % smaller than those in and ). Neurons were transfected with AP180shRNA together with EGFP–mHRP. c is a lower magnification of a, and d is a lower magnification of b. Presynaptic terminals (pre) of transfected neurons are either filled with dark DAB reaction products (white letters in a and g) or have the labeling on the plasma membranes only (black arrowheads in b, e, and f). Synapses formed between nontransfected presynaptic terminals and nontransfected postsynaptic terminals (from the same culture) serve as controls (h–j). Synaptic vesicles become noticeably larger and less densely packed in the AP180 KD-presynaptic terminals than in the control. These changes are seen in the presynaptic terminals irrespective of opposing transfected or nontransfected postsynaptic spines (sp) or dendrites (de). en, endosome; au, autophagosome. Scale bar in c is 500 nm and it applies to d. Scale bar in j is 100 nm and it applies to a, b, e–j. k Size distribution of synaptic vesicles from control and AP180 KD-neurons. Data are pooled from DAB-filled and DAB-outlined presynaptic terminals. Total of 254 potential EM micrographs from two cultures were analyzed (control, n = 1,366 synaptic vesicles; AP180 KD, n = 3,160 synaptic vesicles). i shows that the average size of synaptic vesicles in the AP180 KD-neurons is significantly increased. m shows significantly reduced synaptic vesicle density in the AP180 KD-neurons, as assessed by counting the number of synaptic vesicles per unit area (normalized to lm²). n shows the size of presynaptic terminals. o shows significantly reduced SV cluster size. ***p<0.001; **p<0.01. Data represent mean ± SEM. See also