, 2005 and Tank et al., 1988), which have been attributed to propagating dendritic calcium spikes. While regenerative events have been recorded from proximal smooth dendrites both in vivo (Fujita, 1968 and Kitamura and Häusser, 2011) and in vitro (Davie et al., 2008 and Llinás and Sugimori, 1980), the variability of CF calcium transients measured in distal spiny branchlets suggests that calcium spikes may not always

occur at distal sites. The amplitude of the CF calcium signal is modulated by the somatic holding potential (Wang et al., 2000 and Kitamura and Häusser, 2011), by dendritic field depolarization (Midtgaard et al., 1993), by synaptic inhibition of the dendrites (Callaway et al., 1995 and Kitamura and Häusser, 2011), and by the activity of Idelalisib PFs (Brenowitz and Regehr, 2005 and Wang et al., 2000). The mechanisms underlying these modulations remain unknown. Purkinje cells express a high density of P/Q-type (Usowicz et al.,

1992) and T-type AZD6244 mouse (Hildebrand et al., 2009) calcium channels. P/Q-type channels sustain propagating high-threshold dendritic calcium spikes (Fujita, 1968, Llinás et al., 1968 and Llinás and Sugimori, 1980). In contrast, T-type channels are involved in local spine-specific calcium influx during PF bursts (Hildebrand et al., 2009). Purkinje cell dendrites also express a variety of voltage-gated potassium channels, but their roles in the regulation of dendritic calcium electrogenesis are poorly understood (Etzion and Grossman, 1998, Llinás and Sugimori,

1980, McKay and Turner, 2004 and Womack and Khodakhah, 2004). Here, we used random-access no multiphoton (RAMP) microscopy to monitor the calcium transients induced by CF stimulation (CF-evoked calcium transients [CFCTs]) at high temporal resolution to unambiguously distinguish between subthreshold calcium transients and calcium spikes. We show that calcium spike initiation and propagation in distal spiny branchlets are controlled by activity-dependent mechanisms. CFCTs were mapped optically in Purkinje cell smooth and spiny dendrites using RAMP microscopy (Otsu et al., 2008). At repetition rates close to 1 kHz, the peak of Fluo-4 (200 μM) fluorescence transients was well resolved (Figure S1 available online). Using dual indicator quantitative measurements (see Experimental Procedures), we found that the amplitude of the CFCT (Figures 1A and 1B) decreased with distance from the soma (Figure 1C). In individual spiny dendrites, CFCT amplitude decreased linearly as a function of the distance from the parent dendritic trunk (Figure 1D) by −1.4% ± 0.4% μm−1 (±SD) for spines (r = −0.26, p < 0.001; n = 157 of 14 cells), and −1.5% ± 0.4% μm−1 for spiny branchlet shafts (r = −0.36, p < 0.001; n = 114 of 14 cells). In proximal compartments (<50 μm from soma), fluorescence transients averaged 0.023 ± 0.008 ΔG/R (±SD) in spines (n = 15, 5 cells), 0.020 ± 0.008 ΔG/R in spiny branchlets (n = 19, 7 cells), and 0.014 ± 0.008 ΔG/R in smooth dendrites (n = 25, 10 cells).