To test if intrinsic or input-related differences underlie the difference in simple spike frequency, we recorded PC activity in conditions of limited or no synaptic input. (A) PC activity was recorded in vitro (n = 107 cells, 15 mice) under complete block of synaptic inputs. (B) Spiking frequency in vitro (red) was lower than that in vivo (black) over the range of lobules, but the shape of the curve was similar (r = 0.916, p=0.010, Pearson's correlation). (C1–3) To verify the correlation with zebrin, we recorded activity of EAAT4/zebrin-positive and negative PCs in slices of EAAT4-eGFP mice. Both in lobules II–V (Z+: n = 7 cells, Z−: n = 4; 3 mice; t = 2.910, p=0.017) and lobules VIII-IX (Z+: n = 6, Z−: n = 5; 2 mice; t = 2.352, p=0.043) the difference in simple spike firing frequency was present, further confirming the link with zebrin. (D) Next, extracellular recordings were made in vivo in a6-Cacna1a and PC-Δγ2 mutant mice that have minimized excitatory and no synaptic inhibitory inputs to their PCs, respectively. (E) PC activity in Z+ lobule X of both mutants was lower than that in the predominantly Z− lobules I–III (wild types, lobules I–III: n = 43 cells, 18 mice, lobule X: n = 32 cells, 25 mice, t = 6.808, p<0.001; a6-Cacna1a, I–III: n = 16 cells, 2 mice; X: n = 11 cells, 2 mice; t = 3.979, p<0.001; PC-Δγ2, I-III: n = 11 cells, 3 mice; X: n = 17 cells, 3 mice; t = 4.876, p<0.001). Inset compares the absolute differences in firing frequency between lobules I–III and X. (F) CV2 values of Z− and Z+ SS activity from in vitro recordings (lobules I–III and X: both p<0.001) and in vivo recordings of both a6-Cacna1a mutants (lobule I–III: t = 5.613, p<0.001; lobule X: t = 2.062, p=0.046) and PC-Δγ2 mutants (lobules I–III and X: both p<0.005) were significantly lower than the wild type recordings. Abbreviations: cf, climbing fiber; GC, granule cell; IO, inferior olive; MLI, molecular layer interneuron; PC, Purkinje cell; pf, parallel fiber. Error bars represent SD, *p<0.05, **p<0.001.