Cerebellar LTD in Alert Mice Figure 4. Experimental design and electrophysiological responses during the collision testing. (A) Diagram of the neural pathways concerned in the direct stimulation (Stim.) applied on the top of the parallel fiber (PF). This stimulation produced a negative LFP (superimposed traces on the right corner) recorded by a microfiber placed in the dendritic tress of Purkinje cells (PC). The small arrows indicate the propagation of the orthodromic action potentials (black arrows) and the antidromic action potentials (red arrows) producing collision (red star). The peripheral input coming from the whisker pad is transmitted to the granule cells (GC) via the mossy fiber. (B) Superimposition of the average LFP (n = 10 stimulations) in control (black trace) and during the collision (red trace, red star). The gray vertical arrow indicates the direct stimulation of the PFs while the black vertical arrow indicates the peripheral stimulation of the whisker pad. (C) Evolution of the N2 (blue lines) and N3 (red lines) amplitudes during the collision testing with respect to the control situation recorded before (control 1) and after the collision (control 2). doi:10.1371/journal.pone.0036184.g004 SS and CS during the 8-Hz stimulation protocol To identify more precisely the PC output signals during the 8- Hz stimulation protocol, we analyzed PC recordings (n = 10 PCs in 5 mice) in which both SS and CS responses remained visible, despite the high-frequency stimulation (Fig. 9). We found that the SS frequency doubled from 38.2630.0 Hz before the 8-Hz stimulation to 73.7629.3 Hz during the stimulation (p,0.0001). The evoked CS frequency increased from 0.860.3 Hz before the 8-Hz stimulation to 5.261.5 Hz during the stimulation (p,0.0001). Moreover, during the 8-Hz stimulation, the CS occurred at a mean latency of 20.466.5 ms. Discussion This study demonstrated the existence of an LTD in the cerebellar cortex of alert mice. This LTD was characterized by a specific decrease in the N3 amplitude accompanied by an increase in the latency peaks of the N2 and N3 LFP components. Origins of the N2 and N3 The origin of the LFP components exhibiting the reported plasticity can be approached by the in-depth recording showing a polarity inversion of N3 at the PC level. This result may indicate that this component is produced by superficial dendrites of the PC depolarized by extracellular current flow from the passive sources in the deeper dendrites and PC soma (dipoles vertically oriented, negativity up/positivity down) as proposed by Eccles et al. [31]. Indeed, this specific inversion, demonstrated for the first time in alert animals following peripheral stimulation, was previously demonstrated with electrical stimulation of the juxtafastigial region in the anesthetized cat (see Fig. 10 I in Eccles et al. [23]) and PLoS ONE | www.plosone.org 7 April 2012 | Volume 7 | Issue 4 | e36184