How is synapse number regulated? We have determined the identity of a secreted astrocyte factor that increases the number of synapses made by purified CNS neurons 3-5 fold (Christopherson et al, 2003 SFN abstract). Surprisingly these synapses are not active as measured by patch-clamp recordings of spontaneous mEPSCs. To determine why the synapses are not active, we measured presynaptic function first by measuring the number of synaptic sites that recycle vesicles using an antibody to the luminal domain of synaptotagmin. We found that synapses induced by this astrocyte factor do recycle vesicles, and that there are a comparable number of active synapses induced by this factor and by astrocyte conditioned media. We also investigated the kinetics of presynaptic vesicle recycling using the styryl dye FM1-43. We found that synapses induced by either the purified factor or astrocyte-conditioned media have similar kinetics. To determine if the synapses on these neurons are postsynaptically active, we measured the...