Figure 3. Depth profile analysis of the LFP induced by electrical stimulation. (A) LFP recorded in the molecular layer (Mol. Layer). N1, N2, and N3 components are indicated. (B) LFP recorded near the PC layer where simple spikes (SS) were recorded. At this level, indexed by the occurrence of PC firing, the N3 amplitude decreased. (C) LFP recorded just beneath the PC layer showing the polarity inversion of the N3. Black arrow indicates electrical whisker stimulation. Vertical dotted line indicates N3 latency along different depths. doi:10.1371/journal.pone.0036184.g003 the N3 component while N2 and N1 remained unchanged (Fig. 4C). N3 decrement could be produced by PF–PC synapse blockade or by collision along the PF while N2 associated with the ascending axon–PC synapse, and the granular and Golgi cell activity remain intact. 8-Hz stimulation of the whisker pad had maximal effects on postsynaptic evoked components Different stimulation frequencies induced different plastic changes in the cerebellum [30]; therefore, we tested the immediate effects of whisker stimulation at different frequencies on the N2 and N3 components. On a separate group of mice, involved only in this testing (n = 10), we applied various stimulus trains (30 stimuli per train) with frequencies between 0.2 and 50 Hz. Amplitudes and latencies of the postsynaptic components were obtained by averaging responses to the last 20 stimuli of each frequency train. Figure 5A shows the representative average for each stimulation frequency with the changes observed in the N2 and N3 components in one alert animal. The amplitudes of N2 and N3 decreased with increasing stimulation frequencies, reaching minimum values at frequencies between 8 and 20 Hz. N3 showed an increase in the peak latency (arrowheads in Fig. 5A). In contrast, N1, which reflected the presynaptic input that originated from the mossy fiber in the granule cell layer, showed stable amplitude and latency with stimulation trains between 0.2 and 8 Hz; this result indicated that there were no significant extracerebellar modifications of the input signals. These points are illustrated by the superimposition of the average LFPs from different frequency stimulations in a different animal (ranging from the control situation to 8 Hz; Fig. 5B). The effects on N2 and N3 appeared early in the stimulation train, but they were not sustained beyond the short-duration train (Fig. 5C, after); thus, they did not correspond to plastic changes: i.e., after the 30th Cerebellar LTD in Alert Mice stimulus in the train, a control stimulation induced N2 and N3 peaks similar to those observed in the first stimulus. To quantify the effects of frequency stimulation on postsynaptic components, absolute amplitude and changes in amplitude and latency were assessed for stimulations of 0.2, 0.5, 1, 3, 5, and 8 Hz in 10 animals. In this frequency range, N1 remained stable (Fig. 6A). We therefore used the N1 amplitude as a reference for studying changes in other components. The changes in N2 and N3 amplitudes were significantly different at 1, 3, 5, and 8 Hz compared to N1 (p,0.05; n = 10) (Fig. 6B). However, N2 and N3 showed similar changes in amplitude. Latency changes were calculated as the difference between the peak time under control conditions and at each stimulation frequency. N2 and N3 latency changes increased with increasing frequencies; at 0.5–8 Hz and at 1–8 Hz (p,0.05; n = 10), respectively, they were significantly different from the N1 latencies (Fig. 6C). No significant differences in latency were observed between N2 and N3. In the remaining analyses, we adopted the 8-Hz stimulation protocol for inducing plasticity. Whisker stimulation at 8 Hz induced LTD of postsynaptic components The LTD protocol comprised an 8-Hz electric stimulation of the whisker pad delivered over 10 min in the alert animal. To explore eventual changes in the presynaptic and postsynaptic components, we continuously recorded the LFPs induced by single electrical stimulations (every 1063 s) before (15 min) and after (30 min) the protocol. Figure 7 shows the evolution of LFPs recorded near the PC layer of the Crus II area in response to electrical whisker stimulation in one alert mouse. The traces in Figure 7A represent single-trial LFPs induced by single electrical stimulations before (15 min, Fig. 7A, top), during (10 min, 8 Hz; Fig. 7A, middle), and immediately after the 8-Hz stimulation protocol (30 min, Fig. 7A, bottom). After the 10-min 8-Hz stimulation, N2 and N3 peak latencies increased compared to control conditions. In addition, a marked decrease was observed in the N3 amplitude (Fig. 7B). Mean LFP changes were quantified in 15 animals by comparing averages over each 5-min interval before and after 8-Hz stimulation (Fig. 8A, B). The N3 amplitude was significantly decreased compared to the control condition (p,0.05; n = 15). This effect was maintained during the 30 min of recordings, with a maximum (,50%) observed just after the 8-Hz stimulation trains (Fig. 8A, red triangles). In contrast, no significant changes in amplitudes were observed for the N1 or N2 components (Fig. 8A, black circles and blue squares, respectively). N2 and N3 showed significant latency increases compared to control (p,0.05; n = 15). These latency changes were maintained during the 30 min of recordings, with a maximum (,300 ms) observed just after the 8- Hz stimulation trains (Fig. 8B, red triangles and blue squares). The N1 latency showed a small but significant change just after 8-Hz stimulation (p,0.05; n = 15); however, in contrast to N2 and N3, the N1 latency recovered to control values after the first 5 min of recording (Fig. 8B, black circles). 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 (Fig. 8C) or latency (Fig. 8D) compared to the control condition. To verify the specificity of the reported LTD effects, we used 0.5-Hz stimulation in place of the 8-Hz stimulation protocol. In this case, the N3 LTD effects on both latency and amplitude were absent. PLoS ONE | www.plosone.org 6 April 2012 | Volume 7 | Issue 4 | e36184