Current and Calcium Responses to Local Activation of Axonal NMDA Receptors in Developing Cerebellar Molecular Layer Interneurons Be´ ne´ dicte Rossi¤, David Ogden, Isabel Llano, Yusuf P. Tan, Alain Marty, Thibault Collin* Laboratoire de Physiologie Ce´re´brale, CNRS-UMR 8118, Universite´ Paris Descartes, Universite´ Paris Diderot, Paris, France Abstract In developing cerebellar molecular layer interneurons (MLIs), NMDA increases spontaneous GABA release. This effect had been attributed to either direct activation of presynaptic NMDA receptors (preNMDARs) or an indirect pathway involving activation of somato-dendritic NMDARs followed by passive spread of somatic depolarization along the axon and activation of axonal voltage dependent Ca2+ channels (VDCCs). Using Ca2+ imaging and electrophysiology, we searched for preNMDARs by uncaging NMDAR agonists either broadly throughout the whole field or locally at specific axonal locations. Releasing either NMDA or glutamate in the presence of NBQX using short laser pulses elicited current transients that were highly sensitive to the location of the spot and restricted to a small number of varicosities. The signal was abolished in the presence of high Mg2+ or by the addition of APV. Similar paradigms yielded restricted Ca2+ transients in interneurons loaded with a Ca2+ indicator. We found that the synaptic effects of NMDA were not inhibited by blocking VDCCs but were impaired in the presence of the ryanodine receptor antagonist dantrolene. Furthermore, in voltage clamped cells, bath applied NMDA triggers Ca2+ elevations and induces neurotransmitter release in the axonal compartment. Our results suggest the existence of preNMDARs in developing MLIs and propose their involvement in the NMDA-evoked increase in GABA release by triggering a Ca2+-induced Ca2+ release process mediated by presynaptic Ca2+ stores. Such a mechanism is likely to exert a crucial role in various forms of Ca2+-mediated synaptic plasticity. Citation: Rossi B, Ogden D, Llano I, Tan YP, Marty A, et al. (2012) Current and Calcium Responses to Local Activation of Axonal NMDA Receptors in Developing Cerebellar Molecular Layer Interneurons. PLoS ONE 7(6): e39983. doi:10.1371/journal.pone.0039983 Editor: Fabien Tell, The Research Center of Neurobiology-Neurophysiology of Marseille, France Received April 3, 2012; Accepted May 30, 2012; Published June 27, 2012 Copyright: 2012 Rossi et al. 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 supported by Agence Nationale de la Recherche (contract BLAN08-2_31083), the EU Strep ‘Photolysis’ (LSHM-CT-2007-037765), and Fondation pour la Recherche Me´dicale (FRM Team, A.M.). 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: thibault.collin@parisdescartes.fr ¤ Current address: Life Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, California, United States of America Introduction In the central nervous system, postsynaptic NMDARs are often seen as canonical coincidence detectors in induction of synaptic plasticity. In addition, several lines of evidence indicate that NMDARs are present on presynaptic elements arguing for their possible involvement in presynaptic plasticity processes [1,2]. Presynaptic NMDARs (preNMDARs) have been anatom- ically or functionally detected at both GABAergic [3–6] and glutamatergic [7,8] termini (see [9] for review). Moreover, their implication in long-term plasticity has been suggested in various structures including the visual cortex [6], the neocortex [10,11] and the cerebellum [8,12,13]. In the cerebellum, Lonchamp et al. [14] have shown that the preNMDARs expressed in parallel fibers (PFs) could mediate an increase in the frequency of mIPSCs recorded in Purkinje cells. On the other hand, several studies have suggested that MLIs express preNMDARs. Application of exogenous NMDA increases the frequency of miniature IPSCs (mIPSCs) [3,12,15] following an elevation of presynaptic Ca2+ [16]. This signal is thought to be provided by Ca2+ influx through preNMDARs rather than by activation of voltage-dependent Ca2+ channels (VDCCs) [4,17], and to be amplified through a Ca2+-induced Ca2+ release (CICR) process [4]. Two main sources of glutamate have been proposed to activate preNMDARs on MLIs: spillover triggered by an intense stimulation of parallel fibers (PFs) [12,18] or retrograde signaling from Purkinje cell dendrites [4,19]. In cerebellar slices, the tonic activation of preNMDARs [3,15] has been attributed to a spillover mechanism that is likely to be physiologically limited by glial glutamate transporters [15]. Moreover, glutamate spillover was proposed to contribute to long term potentiation of GABAergic synapses through pre- NMDAR stimulation [12] whereas retrograde activation of preNMDARs has been proposed to potentiate GABA release at the MLI-Purkinje cell synapse [4]. Even though activation of NMDARs clearly affects GABA release, it was recently argued that MLIs may not express axonal NMDARs [20,21]. It was suggested instead that NMDARs are exclusively expressed in the somatodendritic domain, and that the depolarization induced by somatodendritic NMDARs is trans- mitted to the axon compartment due to the passive properties of the axon cable, leading to presynaptic VDCC activation [22]. A similar mechanism may apply to other neurons as well, so that the very existence of functional presynaptic NMDARs in brain neurons remains controversial [23]. PLoS ONE | www.plosone.org 1 June 2012 | Volume 7 | Issue 6 | e39983