Materials and Methods Animals Male FVB mice (n = 30), ages 5–8 months, obtained from an authorized supplier (Charles River Laboratories, Wilmington, MA, USA), were used as experimental animals. All animal procedures were approved by the University of Mons Ethics Committee and conducted in conformity with the European Union directive 609/86/EU. Every effort was made to minimize the number of animals and their discomfort. Surgical preparation Animals were prepared for chronic recordings of LFP and PC single-unit activity [22]. Mice were anesthetized with xylido- dihydrothiazin (Rompun , Bayer, 10 mg/kg) and ketamine (Ketalar , Pfizer, 100 mg/kg). Animals were administered an additional dose of xylido-dihydrothiazin (3 mg/kg) and ketamine (30 mg/kg) when they demonstrated agitation or marked increases in respiration or heart rate during the procedure. In addition, local anesthesia (0.5 mL of 20 mg/mL lidocaine and adrenaline [1:80000, Xylocaine , Astra Zeneca]) was administered subcuta- neously during the soft tissue removal. During surgery, two small bolts were cemented perpendicular to the skull to immobilize the head during the recording sessions, and a silver reference electrode was placed on the surface of the parietal cortex. To allow access to the Crus I and II areas in the cerebellum, an acrylic recording chamber was constructed around a posterior craniotomy (262 mm) and covered with a thin layer of bone wax (Ethicon , Johnson & Johnson). Stimulation of the whisker region Facial dermatomes of the whisker regions were stimulated by air puffs or electrical pulses. Whisker regions were electrically stimulated with a pair of small cutaneous needles inserted under the skin (inter-electrode distance 3–4 mm; Fig. 1A). Electrical stimulation consisted of a single square pulse, 0.2 ms in duration and ,2 mA current intensity, delivered by an isolation unit (Isoflex, AMPI, Israel) connected to an analog pulse generator (Master 8, AMPI, Israel). The amplitude of the current was adjusted to avoid overt movements and animal discomfort. LFPs close to the PC layer and PC single-unit activity were recorded in response to single electrical stimuli delivered at semi-random intervals of 1063 s in the whisker region. When tactile stimulation was required, air puffs were applied at the same rate to the whiskers (2.6 bar and 30 ms in duration; Fig. 1A) by an air pressure system (Picospritzer). Paradigm used for inducing LFP plasticity Preliminary experiments were performed to test the direct effect of different frequency trains of stimulation on the LFPs. For this, 30 electrical stimuli were delivered at different frequencies between 0.2 and 50 Hz. After the analysis of the direct effects on the latencies and amplitude of the different LFP component, the 8-Hz stimulation frequency was selected and used for inducing plasticity. An experimental session consisted of a 15-min control situation in which single electrical stimuli were given at intervals of 1063 s in the whisker region. This period was immediately followed by 10 min of 8-Hz stimulation and then by 30 min of control during which the same single stimuli were applied at the same frequency as in the control situation. Additional controls were performed in different animals in which the period of 10 min at 8 Hz was replaced by 10 min at the same frequency as the control situation. Cerebellar LTD in Alert Mice Single-unit and multiple-unit recordings in alert mice Twenty-four hours after anesthesia, alert mice were restrained for the recording session. The dura was removed over the cerebellum to expose the tissue in the recording chamber. Recordings were performed in the Crus I or II area, and the depths of the electrodes were noted. To avoid unnecessary stress for the animals and movement artifacts, recording sessions were performed in a quiet room when animals were awake and calm. Single and multiple recordings were performed with seven, linearly arranged, quartz-insulated, platinum-tungsten fiber mi- croelectrodes (outer and shaft diameters of 80 mm and 25 mm, respectively), with 250-mm inter-electrode spacing (Thomas Recordings ). Each microelectrode was mounted into a stretched elastic rubber tube to enable proper positioning via DC- micromotors (resolution of 0.27 mm). Neural activity signal recordings were filtered at 100 Hz high-pass and 10 kHz low- pass. LFP and unitary electrical activities were stored digitally on a computer after conversion with an analog–digital converter (Power 1401, CED , Cambridge, UK). The recorded data were digitized continuously at 20 kHz. Off-line analysis and illustrations were performed with Spike2 CED software (CED , Cambridge, UK). 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. Recordings were analyzed when a stable signal was present for longer than 60 s. Electrophysiological responses to electrical stimulation in the whisker region were assessed by both the configuration of the LFP (which must show P1-N1-N2-P2-N3 components, Fig. 1D) and by the identification of appropriate PC firing (modulated by spontaneous whisker movements and electrical stimulation). The P1/N1 component was taken to reflect the evoked activity in the mossy fibers of the granule cell (GC) layer. When this component was stable during the experiment, we assumed there was no extracerebellar modification of the input signals. Collision testing between the whisker pad stimulation and direct PF stimulation Stimulation of the PFs was delivered by a bipolar parylene- coated microelectrode placed just below the surface of the cerebellar cortex [21] 0.5 mm laterally from the recording electrode. The position of the bipolar electrode was adjusted to produce a negative field in the superficial part of the molecular layer corresponding to the activity of the PF–PC synapses. Then the presence of stable LFP triggered by the whisker pad stimulation was checked before the application of a single electrical stimulus at the PF. To produce a collision between the afferent volley traveling in the pathway between the GC and the PC, and the antidromic volley elicited by the PF stimulation, the latter was applied 2 ms before the whisker pad stimulation. The amplitude of each LFP component was then compared before, during, and after the collision test. Data analysis The peak-amplitudes and latencies of N2 and N3 components were used as an index of the postsynaptic responses [21,23,24]. These were compared with the SS responses of the recruited PCs. Data were analyzed off-line for amplitude and latency quantifi- PLoS ONE | www.plosone.org 2 April 2012 | Volume 7 | Issue 4 | e36184