Figure 1. Experimental design and electrophysiological re- sponse to electrical stimulation of mouse whiskers. (A) Animals were prepared for chronic recordings of local field potentials and unitary extracellular activity in the Purkinje cell layer of the Crus I/II area. Facial dermatomes of the whisker region were electrically or tactilely stimulated with a pair of needles under the skin (Stim.) or an air puff Cerebellar LTD in Alert Mice pulse, respectively. Sensory information comes into the Crus I/II area from the trigeminal nucleus (Tn) in the brainstem, which receives afferent signals from the trigeminal ganglion (Tg). The LFP recorded in the alert animal induced by tactile stimuli consisted of two major negative waves corresponding to trigeminal (T) and cortical (C) responses (upper trace). (B) In some of the recordings, the T component appeared as two separate components (N2 and N3). The figure shows superimposed LFPs (upper trace, n = 7) and the corresponding mean average (with error bars in gray at bottom) for the LFP acquired after tactile stimulation of the whisker. (C) Superimposed LFPs (upper trace, n = 7) and the corresponding mean average (with error bars in gray at bottom) for the LFP acquired at the same recording place shown in B but after electrical stimulation of the whisker. 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. (D) The lower trace shows T-related components enlarged from a representa- tive local field potential recording from the Crus II area in the Purkinje cell layer in response to a single-pulse electrical stimulation of the whisker pad (Stim. trace). Early response associated with sensory input in the cerebellum via the trigeminal nucleus is characterized by P1-N1- N2-P2-N3 components. doi:10.1371/journal.pone.0036184.g001 cations of N1, N2, and N3. For this analysis, 30 successive evoked field potentials were averaged to obtain one average data point every 5 min. The amplitude was computed by peak-to-peak measurements. For this calculation, negative peaks were compared to the trough of the preceding positive wave. When the P1 positive peak was not evident, we used the inflection point observed in the averaged trace for the N1 measurement. For N3 measurements, the amplitude was set as the difference between the positive peak between N2 and N3 and the negative peak of N3. Latency was determined as the time difference between stimulus onset and N1, N2, or N3 averaged peaks. For comparisons between animals, amplitude values for each one of the components of the LFP were normalized for statistical analysis. Discrimination between PC simple and complex spikes was performed with Spike2 CED software (CED, Cambridge, UK) and controlled visually before analysis. Waveform averaging was performed with the corresponding function of this software on a 120-s minimum recording. The SS firing response was evaluated on an averaged histogram that included all the stimulations during the recording of a given cell (bin size = 1 ms). The significance of the response was evaluated by comparing the averaged spike number per bin on five successive bins following stimulation to the bin values in the interval (2200 to 2100 ms) before stimulation. The effect of a single stimulation on PC firing was also analyzed by superimposing single trace trials, which allowed the identification of evoked simple and complex spikes. A CS firing response was defined as the occurrence of a CS during the interval (10–50 ms) following at least 30% of the stimulations [25]. The student’s t test and one-way ANOVA for repeated measures were performed in SPSS (v 14.0, SPSS Inc ). Statistical significance was set at p,0.05. Unless indicated otherwise, the results are shown as mean6SEM. Results The electrophysiological response to electrical and tactile whisker stimulation near the PC layer of the Crus I or Crus II area (at 1063 s intervals) was characterized in the alert mice. The LFP recorded in the alert animal induced by tactile stimuli consisted of two major negative waves (Fig. 1A). These two components have been previously reported in anesthetized rats as T and C, corresponding to trigeminal and cortical responses [20,26,27]. In the present condition, T and C components peaked PLoS ONE | www.plosone.org 3 April 2012 | Volume 7 | Issue 4 | e36184