at 4.563.5 ms and 16.169.4 ms (mean6SD; n = 5), respectively. In some of the performed recordings, the T component appeared as two separated components (Fig. 1B). In these instances, the latency of the two peaks was 2.660.5 ms and 3.860.5 ms and 12.463.4 ms for the C component (mean6SD; n = 5). To reduce the variability of the LFP observed after tactile stimulation by an air-puff pulse in alert mice, we tested subcutaneous electrical stimulation of whiskers. This alternative sensory stimulation protocol allows a diminishing of the duration of the stimulus (0.2 ms vs 30 ms for air pulse), increasing the reproducibility of the responses. Figure 1 shows the superimposed LFPs (n = 7) and the corresponding mean average (with error bars in gray) for the LFP acquired at the same recording location after tactile (B) and electrical stimulation of the whisker (C). A major reproducibility of the T-related components in the superimposed traces and a decrement in the error bars in the mean average of LFP were observed after electrical stimulation in comparison to air puff stimulation. In consequence, electrical stimulation of whiskers was used to characterize plastic changes in the cerebellum of the alert mice. Presynaptic and postsynaptic components of the LFP LFP recorded near the PC layer after electrical stimulation of the whisker comprised three well-defined waves appearing before the slower C component (Fig. 1D). In this experiment, only T- related components were analyzed because of their direct relation with sensory input in the cerebellum via the trigeminal nucleus [27]. These three early negative waves, designated here as N1, N2, and N3, peaked at 1.860.4 ms, 3.360.3 ms, and 4.560.2 ms (mean6SD; n = 15), respectively, after stimulus onset. The mean peak-to-peak amplitude of these components corresponded to 0.1960.15 mV, 0.4360.18 mV, and 0.4860.19 mV, respectively (mean6SD; n = 15). The short latency and small amplitude of N1 suggested a correspondence with the classical P1–N1 presynaptic input component [23] originating in the mossy fiber in the GC layer, originally documented following juxtafastigial stimulation in anesthetized cats. Additional experiments were required to clarify the nature of the later N2 and N3 components. To this effect, we attempted different approaches, namely (1) PC unitary extracel- lular recording, (2) depth profile analysis of the induced field, and (3) collision testing between whisker stimulation and direct PF stimulation. To characterize the relationship between the potentially postsynaptic components of the LFP recorded near the PC layer and the firing behavior of PCs, single electrical stimuli were applied to the whisker pad (at 1063 s intervals) during extracellular unitary recording of the identified PC. A total of 45 identified PCs were included in the analysis. A neural signal was considered to originate from a PC when it presented two types of spiking activities: SS, characterized by a single depolarization (300–800 ms) that occurred between 20 and 200 Hz; and CS, characterized by an initial fast depolarization (300–600 ms), followed by smaller and relatively constant wavelets. Simple and complex spikes were considered to originate from the same PC when a transient pause (,15 ms) in SS firing followed each CS (Fig. 2A,D,E asterisks). PC response to electrical whisker stimulation consisted of SS and CS generation. In Figure 2B, superimposition of single trials (n = 16) showed spontaneous SS firing before the stimuli and SS and CS evoked by electrical stimulation of whiskers (Stim in Fig. 2B). Of interest, SS occurred with the same latencies as the above-mentioned N2 and N3 components (3.5–4.5 ms, in this case; Fig. 2B) whereas the CS occurred 9–13 ms after the stimulation. For the majority of the Cerebellar LTD in Alert Mice recorded PCs, a very short silent period of 3–4 ms occurred after the early SS response and preceded the arrival of the CS. The histogram (Fig. 2C, top) shows the initial SS increase followed by a CS increase (Fig. 2C, bottom); this finding confirmed the consistent temporal relationship between the peripheral stimulation and these early SS responses. A later inhibition occurred between 24 and 100 ms after the stimulus. In Figure 2, superimposition of the same PC recording triggered by CS (dotted line) is shown during spontaneous firing (n = 43, Fig. 2D) or in response to electrical whisker stimulation (n = 58, arrowheads in Fig. 2E). One of the traces is shown in red to facilitate identification of the SS and CS. This approach, applied to each of the recorded cells, allowed us to confirm that the spontaneous CS originated from the same PC as the CS evoked by stimulation. Therefore, the PC response was characterized by a short excitation (SS+CS) followed by a longer inhibition, with a variable duration (20 ms to 150 ms). In the analyzed PC population (n = 45), the onset of the first evoked spike occurred at a mean latency of 3.060.5 ms (Fig. 2F, black bars) whereas the onset of the second group of spikes had a mean latency of 5.261.2 ms (mean6SD; measured in 45 PCs) (Fig. 2G, gray bars). The first two peaks observed in the distribution of SS latencies measured in the PC population corresponded to the time of the second and third LFP components, respectively. For the same PC population, the CS onset always occurred after the evoked SS, at a mean latency of 14.865.1 ms (Fig. 2F). Therefore, results from PC spikes recording after whisker electrical stimulation strongly suggest that N2 and N3 waves are associated with postsynaptic components. Although SS activity from PCs was closely associated with the N2 and N3 components, the contribution of GC and Golgi cell placed a few micrometers beyond the recording site must not be discarded. Furthermore, additional in-depth penetrations were performed to better identify the postsynaptic nature of the N2 and N3 components. When the microfiber penetrated the molecular layer perpendicularly, the negative polarity of the postsynaptic compo- nent was maintained until the PC layer was approached (Fig. 3A). At this level, indexed by the occurrence of PC firing, the N3 amplitude decreased (Fig. 3B). Then, continuing to move in depth, just beneath the PC layer, the postsynaptic field suddenly was changed into a positive component peaking at the exact latency as the previous negative peak (Fig. 3C). When such an inversion occurred, it could be repeated and reversed again by moving the microfiber up and down in the same configuration. Of note, we have observed such an inversion only for the N3 component and not for the N1 and N2 components. This pattern indicates that the postsynaptic generator of N3 is produced by PC dipoles vertically oriented with the negative pole situated in the superficial part of the dendrite arborization [28]. The nature of N2 is more complex and could correspond to the granular and Golgi cell activity [23] directly followed by the action of an ascending axon–PC synapse [29]. Finally, to check for the postsynaptic origin of the N2 and N3 components, we electrically stimulated the PF to partly block the afferent volley (elicited by the whisker pad stimulation) traveling to the PC via the ascending axons of the granule cells and the PF beam (Fig. 4A). Prior to the collision testing, we first demonstrated that the PF stimulation could induce a postsynaptic LFP at the PC level. This stimulation produced a negative field in the molecular layer, peaking at a latency of about 2.560.4 ms (mean6SD; n = 10) for a stimulation distance of about 0.5 mm (superimposed traces in Fig. 4A). Then a collision test between whisker pad and the PF stimulation given 2 ms earlier was performed in different animals (n = 5) (Fig. 4B). This test induced a significant decrease in PLoS ONE | www.plosone.org 4 April 2012 | Volume 7 | Issue 4 | e36184