Synaptic vesicle tethers to the AZ. For proximal vesicles (within 45 nm from the AZ, as virtually no vesicles were tethered in more distal areas). (A) Number of tethered vesicles per synapse, which was strongly reduced in RIM1α KO-altered synapses. (B) Fraction of proximal vesicles tethered to the AZ. (C) Histogram of number of tethers per proximal vesicle. Note that no vesicles with more than two tethers were found in KO-altered synapses. (top) The cartoon represents the bins of the histogram: nontethered vesicles (left), vesicles with one to two tethers (middle), and vesicles with multiple tethers (right). AZ (gray), proximal vesicles (yellow), and tethers (blue) are shown. (D) Number of tethers per AZ unit area. (E) Tether length. (F) Histogram of tether lengths. Short tether formation was impaired in RIM1α KO synapses. A, D, and E show mean values and SEMs (error bars). B, C, and F show number of occurrences (consequently no error bars are displayed). Confidence values: *, P < 0.05; **, P < 0.01. The numbers of animals, synapses, vesicles, and tethers analyzed for each category are shown in Table S1. SV, synaptic vesicle.