Cerebellar LTD in Alert Mice Figure 5. Effects of stimulation frequency on evoked components. (A) Representative responses of one mouse to various stimulus trains (30 stimuli per train) at frequencies between 0.2 and 50 Hz (indicated at left). Each trace shows the mean local field potential (averaged over the last 20 stimuli) recorded near the Purkinje cell layer of the Crus I/II area. In control conditions, the stimuli were delivered every 1063 s. To facilitate visual comparison, dotted lines have been placed to indicate the locations of the N1, N2, and N3 peaks observed before stimulation. The arrowheads indicate shifts in the peak latencies. (B) Superimposed traces of mean averaged local field potentials obtained at stimulation frequencies from control to 8 Hz. Traces were aligned with the stable N1 component. Dotted lines indicate the locations of the N1, N2, and N3 peaks observed before stimulation. (C) Single-trial traces that correspond to the local field potentials in response to the first 10 stimuli (8 Hz) in a train (numbered at left). Single control stimuli responses were recorded just before the train (top) and just after (bottom) the 10th stimulus (8 Hz). doi:10.1371/journal.pone.0036184.g005 during cutaneous electrical stimulation of the snout in decerebrate and pentobarbitone-anesthetized rats [32]. Moreover, the physi- ological origin of the present N3 is reinforced by the collision test demonstrating a specific decrease of the N3 component when the PFs were directly stimulated just before (,2.0 ms) the whisker pad stimulation. Similarly, the N3 evoked by the juxtafastigial stimulation was also suppressed by a concomitant PF stimulation in the cat [23]. This result suggests that the sensory input contributing to the N3 travels along the PF beam and that N3 reflects the activity of PF–PC synapses. We note here that although the assumption that PF stimulation effects are caused by collision along the pathway from the GC to the PC via the PF, additional effects induced by other elements of the cerebellar cortex cannot be ruled out. Nevertheless, the collision test finding was also reinforced by the timing of the SS synchronized on the N3 field. A similar conjunction has already been illustrated in Figure 9A in Eccles et al. [23]. At the same time, the fact that N2 is not modified by either the collision or depth inversion points to a different site of generation. N2 could possibly reflect the conjoint activity from the granule and Golgi cells, corresponding to the N2 component recorded in vitro and in anesthetized preparation [20,30], and/or the synapse between the ascending segment of the GC and PC at the soma level. Although our results have established that the evoked SS and the N2 and N3 components occurred simultaneously, the exact nature of the involved excitatory synapses remains to be clarified. Two non-exclusive possibilities can be considered. First, the early evoked SS may arise from activation of the PF–PC synapse; second, it could result from a synapse between the ascending portion of GC axons and the PC dendrites. The latter hypothesis is PLoS ONE | www.plosone.org 8 April 2012 | Volume 7 | Issue 4 | e36184