Figure 6.

Synapsin IIa affects the size of the RP but not the RRP. FM4-64 unloading was used to assess the effect of reintroduction of synapsin IIa on the size of the RRP and the RP of TKO neurons. A, Representative image of presynaptic terminals of a TKO neuron loaded with FM4-64 by 1200 stimuli delivered at 10 Hz (red, left). These terminals belong to a neuron expressing GFP-synapsin IIa (green, right). B, Example of measurement of FM4-64 unloading. Unloading of FM4-64 by 40 stimuli delivered at 20 Hz served to unload FM4-64 from the RRP, whereas two successive trains of 1800 and 3000 stimuli delivered at 10 Hz subsequently unloaded dye from SVs remaining in the RP. The sum of both signals served as our measure of the total recycling pool. C, Determination of the size of the total recycling pool. Distribution of the magnitude of FM4-64 fluorescence loss during complete unloading (left), cumulative probability derived from the same data (center), and average of the median value determined for each image (right). The median size of the total recycling pool was significantly reduced in TKO terminals compared with TWT terminals. Likewise, reintroduction of synapsin IIa into TKO terminals partially rescued its size (*p < 0.05). D, E, Determination of the size of the RRP and the RP. Deletion of all synapsins had no effect on the size of the RRP (D), but decreased the size of the RP (E). Reintroduction of synapsin IIa into TKO neurons did not affect the RRP, but partially rescued the RP (*p < 0.05). Measurements were performed from 350–1200 synapses in each image of TWT (n = 8), TKO (n = 8), or TKO neurons expressing synapsin IIa (n = 9).