Sensory Stimulation-Dependent Plasticity in the Cerebellar Cortex of Alert Mice Javier Ma´ rquez-Ruiz1, Guy Cheron2,3* 1 Divisio´ n de Neurociencias, Universidad Pablo de Olavide, Sevilla, Spain, 2 Laboratory of Electrophysiology, Universite´ de Mons, Mons, Belgium, 3 Laboratory of Neurophysiology and Movement Biomechanics CP601, Universite´ Libre de Bruxelles, Brussels, Belgium Abstract In vitro studies have supported the occurrence of cerebellar long-term depression (LTD), an interaction between the parallel fibers and Purkinje cells (PCs) that requires the combined activation of the parallel and climbing fibers. To demonstrate the existence of LTD in alert animals, we investigated the plasticity of local field potentials (LFPs) evoked by electrical stimulation of the whisker pad. The recorded LFP showed two major negative waves corresponding to trigeminal (broken into the N2 and N3 components) and cortical responses. PC unitary extracellular recording showed that N2 and N3 occurred concurrently with PC evoked simple spikes, followed by an evoked complex spike. Polarity inversion of the N3 component at the PC level and N3 amplitude reduction after electrical stimulation of the parallel fiber volley applied on the surface of the cerebellum 2 ms earlier strongly suggest that N3 was related to the parallel fiber–PC synapse activity. LFP measurements elicited by single whisker pad stimulus were performed before and after trains of electrical stimuli given at a frequency of 8 Hz for 10 min. We demonstrated that during this later situation, the stimulation of the PC by parallel and climbing fibers was reinforced. After 8-Hz stimulation, we observed long-term modifications (lasting at least 30 min) characterized by a specific decrease of the N3 amplitude accompanied by an increase of the N2 and N3 latency peaks. These plastic modifications indicated the existence of cerebellar LTD in alert animals involving both timing and synaptic modulations. These results corroborate the idea that LTD may underlie basic physiological functions related to calcium- dependent synaptic plasticity in the cerebellum. Citation: Ma´rquez-Ruiz J, Cheron G (2012) Sensory Stimulation-Dependent Plasticity in the Cerebellar Cortex of Alert Mice. PLoS ONE 7(4): e36184. doi:10.1371/ journal.pone.0036184 Editor: Izumi Sugihara, Tokyo Medical and Dental University, Japan Received December 16, 2011; Accepted April 3, 2012; Published April 26, 2012 Copyright: 2012 Ma´rquez-Ruiz, Cheron. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was funded by the Belgian Federal Science Policy Office, the European Space Agency, (AO-2004,118), the Belgian National Fund for Scientific Research (FNRS), and the research funds of the Universite´ Libre de Bruxelles and the Universite´ de Mons (Belgium). This work was also supported by the Ministerio de Educacio´ n y Ciencia Programa Nacional de Movilidad de Recursos Humanos del Plan nacional de I-D+I 2008-2011 of the Spanish Government. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: gcheron@ulb.ac.be Introduction The first experimental evidence of long-term depression (LTD) at the parallel fiber (PF)–Purkinje cell (PC) synapse was induced by conjunctive activation of parallel and climbing fiber (CF) inputs in decerebrated rabbit [1] and in slices [2]. This evidence was consistent with the Marr-Albus-Ito models of cerebellar learning [3–5]. Subsequently, other types of cerebellar plasticity have been demon- strated [6]. Presynaptic [7] and postsynaptic [8] long-term potentiation (LTP) at the PF–PC synapse supported the concept of bidirectional plasticity controlled by intracellular Ca2+ signaling [9,10]. When CFs were stimulated at 5 Hz, the CF–PC synapses underwent LTD [11]. Exploration studies of the cellular and molecular mechanisms implicated in LTD at the PF–PC synapses have clarified the involvement of the metabotropic glutamate receptor subtype 1 [12], protein kinase C [13], and clathrin-mediated internalization of postsynaptic AMPA receptors [14]. Other studies have shown that the activation of the postsynaptic a-calcium/calmodulin- dependent protein kinase II and the nitric oxide–cyclic GMP- protein kinase G cascade [15] were also involved in LTD. In addition, LTD was associated with both presynaptic [16] and postsynaptic [17] NMDA receptors. PF–PC LTD and LTP have been confirmed in decerebrate animals [18,19]. LTD and LTP were also shown to occur in the granular cell layer following facial tactile stimulation in the anesthetized rat [20]. That study further demonstrated that changes in local field potential (LFP) amplitudes were accompa- nied by changes in latency, which indicated a possible role of plasticity in the spatio-temporal spike sequences of the cerebellar input. Of interest, Wang et al. [21] demonstrated a PF–PC LTP following high-frequency stimulation (100 Hz) in the PF of anesthetized mice. However, to date, no work had demonstrated either LTP or LTD in alert animals. Here, we investigated the existence of LTD plasticity in alert mice by using electrical stimulation of the whisker pad. We found that LTD occurred in the N3 LFP component (amplitude decrease and delayed latency), after 10 min of whisker stimulation at a frequency of 8 Hz. The LFP N2–N3 component coincided with the early evoked simple spikes (SS) of the PC and was followed by an evoked complex spike (CS); this conjugated activation of the PC was reinforced during the 8-Hz stimulation. Our results provide the first demonstration that LTD can be induced in the cerebellum of alert animals in response to sensory peripheral input. PLoS ONE | www.plosone.org 1 April 2012 | Volume 7 | Issue 4 | e36184