Figure 6. Changes in amplitude and latency of evoked local field potential components along the whisker stimulation frequency range. (A) Absolute amplitude of postsynaptic compo- nents at stimulations of 0.2, 0.5, 1, 3, 5, and 8 Hz. Changes in mean amplitudes (B) and latencies (C) compared to control conditions were induced by stimulation frequencies of 0.2, 0.5, 1, 3, 5, and 8 Hz. N1 (black circles), N2 (blue squares), and N3 (red triangles) components are expressed as mean6SEM. Significant differences (asterisks) were evaluated for the postsynaptic components (N2 and N3) with respect to the presynaptic input (N1). (p,0.001, ***; p,0.01, **; p,0.05, *). doi:10.1371/journal.pone.0036184.g006 in accordance with Llina´s [29], who suggested that the ascending portion of GC axons provides the major granular input into the PC. Moreover, the synapses along the ascending segments have a Cerebellar LTD in Alert Mice higher release probability [33], but the large majority of the PF– PC synapses do not generate functional links [34]. Recent studies [35] confirm the initial proposal of Llina´s [29] about the vertical (patchy) organization of the PC and demonstrate the existence of two specific excitatory synapses spatially segregated on the PC dendrite. Of interest, the ascending segment synapses are made exclusively on the smallest-diameter PC dendrites and present a vertical orientation roughly perpendicular to that formed by the PF–PC dendrites (horizontal orientation) (Fig. 2 of Lu et al., [35]). According to the present results, these new elements may be interpreted as follows: (1) the earlier SS response may be explained by a shorter pathway (ascending axon to the PC dendrite shorter than ascending axon+PF pathway to PC dendrite), and the ascending axon synapse made on the smallest dendrite could facilitate the earlier recruitment of this PC response; or (2) the perpendicular orientation of the two distinct synaptic inputs may explain why the N3 but not the N2 is reversed during the same in- depth recording. Finally, the ascending axons have been proposed to be mainly hard wired and resistant to LTD [33] and to play the role of event detectors. In contrast, the PF–PC synapses display short- and long- term synaptic plasticity [2,8,9,19]. In this context, the absence of an amplitude decrease in the N2 after the 8-Hz stimulation imposed in this study might indicate support of this LFP component by the ascending axon–PC synapse while the N3 was more directly related to PF–PC synapses. Evidence for cerebellar LTD in alert animals A wealth of evidence supports the notion that the LTD effect demonstrated here corresponds to the cerebellar learning theory [3–6,36]. In that theory, the CF induces LTD at the PF–PC synapses, specifically for the PFs that were active at or just before the occurrence of the CF input. The present paradigm does not require any type of motor learning, and both climbing and mossy fiber inputs are involved. As in the original formulation of this theory, we demonstrated here that whisker stimulation consistently resulted in the conjunctive recruitment of both climbing and mossy fibers, as evidenced by the SS firing increase shortly followed by CS firing. Moreover, we demonstrated that during the 8-Hz stimulation, the timing of this conjunctive recruitment was conserved, and the evoked CS reached a high frequency rate. Although no motor learning task is required in the present sensory stimulation paradigm, the increase of the CS frequency during the 8-Hz stimulation period is reminiscent of the reported increase in CS during the learning period of a motor task [37]. After that pioneering work, the increase in CS frequency during behavioral learning was confirmed [38,39]. Furthermore, the seminal in vitro study of Ito and Kano [2] demonstrated that conjunctive stimulation of PFs and CFs at 4 Hz induces an LTD in the field potential associated with postsynaptic excitation in dendrites of PCs and other cortical cells. Consistent with that evidence, our findings in alert animals indicated that a peripheral stimulation that activated both climbing and mossy fibers could induce LTD in the N3 component. Llina´s et al. [40] promoted an alternative to the view that LTD played a role in learning, hypothesizing that LTD may be neuroprotective against Ca2+-mediated excitotoxicity. In that context, the present 8-Hz stimulation may be viewed as a favorable influence on Ca2+ influx overflow in which a 5-fold increase in the CF inputs triggers a powerful Ca2+ influx [41]. In addition, the concomitant increase in PF input may contribute to a more focal Ca2+ increase [42,43]. Thus, the present LTD in the N3 components might be used as an electrophysiological index of PC neuroprotection. PLoS ONE | www.plosone.org 9 April 2012 | Volume 7 | Issue 4 | e36184