Endocytic vacuoles formed following a short pulse of K+-stimulation contain a plethora of presynaptic membrane proteins

It is now well established that the membrane of synaptic vesicles is recycl ed following exocytosis. However, little is known concerning the identity o f the primary or secondary endocytic structures and their molecular composi tion. Using cultured rat cerebellar granule cells we combined uptake of hor seradish peroxidase as a fluid phase marker and immunogold labeling for a v ariety of presynaptic proteins to assess the molecular identity of the stim ulation-induced endocytic compartments. Short periods (5 or 30 s) of stimul ation with 50 mM KCl were followed by periods of recovery for up to 30 min. Stimulation resulted in the formation of horseradish-peroxidase-filled vac uoles in the axonal varicosities as the apparent primary endocytic compartm ent. Horseradish peroxidase-filled synaptic vesicles were formed when stimu lated cells were allowed to recover in horseradish peroxidase-free culture medium. Horseradish peroxidase-filled vacuoles as wells as vesicles contain ed the synaptic vesicle membrane proteins VAMP II, synaptotagmin, SV2, and synaptophysin, the vesicle-associated proteins rab 3A and synapsin I, and i n addition SNAP-25. No incorporation of vesicle proteins into the plasma me mbrane was observed. Horseradish peroxidase-filled vesicles and vacuoles ge nerated on incubation of unstimulated granule cells with horseradish peroxi dase for prolonged periods of time were equally immunolabeled. Renewed stim ulation of prestimulated granule cells with either 100 mM KCl or 30 mu M Ca 2+ ionophore A23187 resulted in a reduction of horseradish peroxidase-fille d vacuoles suggesting that the vacuolar membrane compartment was exocytosis -competent. Our results suggest that varicosities of cultured cerebellar granule cells possess a fast stimulation-induced pathway for recycling the entire synapti c vesicle membrane compartment. The primary endocytic compartment represent s not a synaptic vesicle but a somewhat larger vesicle protein-containing v acuolar entity from which smaller vesicles of identical protein composition may be regenerated. Endocytic vacuoles and synaptic vesicles share membran e and membrane-associated proteins and presumably also major functional pro perties. (C) 1999 IBRO. Published by Elsevier Science Ltd.