Cerebellar LTD in Alert Mice Figure 8. Time course of the effects induced by an 8-Hz stimulation train on evoked field potential components. (A) Time course of amplitude changes for N1 (black circles), N2 (blue squares), and N3 (red triangles) before (negative time periods) and after (positive time periods) the 8-Hz stimulation protocol (not shown). Mean normalized values were calculated for each 5-min interval of data from 15 alert animals. Data points are mean6SEM. (B) Time course of N1 (black circles), N2 (blue squares), and N3 (red triangles) latency changes before (negative time periods) and after (positive time periods) the 8-Hz stimulation protocol (not shown). Mean data represent the time difference at peak latency between each 5-min interval and the mean value measured in control conditions (negative time periods). Significant differences from control are indicated with asterisks. A small but significant (*) difference observed in the N1 presynaptic component showed recovery to control values after the first 5 min. (C, D) Controls were performed in different animals (n = 10) for which the 10-min period at 8 Hz was replaced by 10 min at the same frequency as the control situation. No significant changes were observed in the amplitude or latency compared to the control condition. (p,0.001, ***; p,0.01, **; p,0.05, *). Data points are mean6SEM. doi:10.1371/journal.pone.0036184.g008 entities recruited by the peripheral stimulus, as demonstrated in the somatosensory cortex of awake mice [53]. In the present study, the first PF–PC excitation, the evoked SS, occurred ,20 ms before the evoked activation of the CF. This temporal link between PF and CF activities may indicate a role similar to that revealed in PC spines, when PF activation occurred 50–200 ms before CF activation [54] and gave rise to supralinear calcium transients. LTD and the timing effect In the context of pattern recognition theory, in vitro and PC model studies [55] have shown that a PF input alone is sufficient to evoke, upon spontaneous SS firing, a highly reproducible pattern of three SS spikes followed by a pause. In that work, the timing of the second and third evoked spikes was delayed after LTD induction, without the contribution of any other neuronal elements in the cerebellar network. This pattern was very similar to what we observed in the present study with trigeminal input. However, we cannot rule out the possibility that the mossy fiber– GC synapses were implicated in the observed time-delay effect. The time shifts were roughly the same for the N2 and N3; thus, this temporal plasticity may have occurred in the GC layer. This alternative has been demonstrated in vitro [30] and in the anesthetized rat [20]; moreover, it gains theoretical support from the notion that the mossy fiber–GC synapses, regulated by Golgi cells, form a template for timing adjustments of the mossy fiber input [56]. Do the molecular inhibitory interneurons contribute to the present LTD? The inhibitory interneurons are excited by both PF [31] and CF inputs [19]; therefore, they might be involved in the present plasticity. The N2 and N3 latency shifts fit relatively well with the PC output spike shift measured in vitro after the induction of an inhibitory postsynaptic potential [50]. However, the contribution of inhibitory synapses to the evoked SS timing does not necessarily imply their participation in LTD. Indeed, the fact that the pairing PLoS ONE | www.plosone.org 11 April 2012 | Volume 7 | Issue 4 | e36184