Aims and Methods: Neighboring Purkinje neurons (PNs) connect each other via recurrent axon collaterals, which form synapses that switch from paired-pulse depression to facilitation (PPF) during postnatal development (Orduz & Llano 2007, Watt et al. 2009). It has been suggested that saturation of the high-affinity Ca²⁺ binding protein calbindin-D28k (CB), which is abun¬dantly expressed in PNs, underlies facilitation (Orduz & Llano 2007). Here we addressed this hypothesis using paired electrophysiological recordings from wild-type (WT) and CB mutant mice combined with two-photon calcium imaging. Results: We confirm that PPF occurs at WT synapses of 2- to 3-week-old mice with a paired-pulse ratio of ~2 at 3 ms interstimulus intervals (ISI). We find that PPF mainly results from a high failure rate of ~45 % in the first release process that declines to ~25 % for the second release (at 3 ms ISI). EPSC amplitudes of responses that followed a failure were only slightly increased by 17 % compared to responses to the first stimulus. Two-photon imaging revealed that the volume-averaged Ca²⁺ signals in WT presynaptic terminals were small and could only be resolved upon repetitive activation. Numerical simulations indicate that the fractional Ca²⁺ occupancy of CB increases from a resting level of ~10 % to a peak of <15 % after the first stimulus. CB mutants showed ~2fold larger peak Ca²⁺ signals. However, PPF was not significantly different from the WT. Conclusion: These findings argue against the hypothesis that saturation of CB is the dominant factor governing PPF at recurrent PN synapses. Supported by the DFG. References: Orduz D & Llano I, 2007, PNAS 104, 17831-17836. Watt AJ, Cuntz H, Mori M, Nusser Z, Sjöström PJ & Häusser M. 2009, Nat Neurosci 12, 463-473.