potentials (20 ms intervals) were produced by depolarizing the cell for 3 ms to 0 mV from a holding value of 270 mV thus inducing a propagated action potential [46]. Whole-cell recording pipettes were filled with (in mM) 140 K- gluconate, 5.4 KCl, 4.1 MgCl2, 9.9 HEPES-K, 0.36 Na-GTP, 4 Na-ATP, pH was adjusted to 7.3 with KOH. For 2-photon laser scanning imaging experiments, this solution was supplemented with 400 mM Fluo 4 (Invitrogen). For single photon excitation, the solution contained 50 mM Oregon Green 488 BAPTA-1 (OGB-1; Invitrogen). and Alexa 488 fluor (20 mM; Invitrogen) was added for local uncaging to facilitates axon identification early in the experiments. However, DF/F0 values reported here for trains of action potentials (DF/F0 = 34.761.3%; n = 15; Fig. 5C) are lower than those previously reported for the axons using OGB-1 alone. For all Ca2+ imaging experiments, neurites identification was confirmed by analysis of action potential induced Ca2+ transients, which are markedly larger in the axon than in dendrites as reported [25]. Analysis was performed by calculating the average fluorescence in regions of interest (ROIs) as a function of time as detailed before [45]. References 1. Duguid I, Sjo¨stro¨m PJ (2006) Novel presynaptic mechanisms for coincidence detection in synaptic plasticity. Curr Opin Neurobiol 16: 312–322. 2. Corlew R, Brasier DJ, Feldman DE, Philpot BD (2008) Presynaptic NMDA receptors: newly appreciated roles in cortical synaptic function and plasticity. Neuroscientist 14: 609–625. 3. Glitsch M, Marty A (1999) Presynaptic effects of NMDA in cerebellar Purkinje cells and interneurons. J Neurosci 19: 511–519. 4. Duguid IC, Smart TG (2004) Retrograde activation of presynaptic NMDA receptors enhances GABA release at cerebellar interneuron-Purkinje cell synapses. Nat Neurosci 7: 525–533. 5. Fiszman ML, Barberis A, Lu C, Fu Z, Erdelyi F, et al. (2005) NMDA receptors increase the size of GABAergic terminals and enhance GABA release. J Neurosci 25: 2024–2031. 6. Lien CC, Mu Y, Vargas-Caballero M, Poo MM (2006) Visual stimuli-induced LTD of GABAergic synapses mediated by presynaptic NMDA receptors. Nat Neurosci 9: 372–380. 7. Berretta N, Jones RS (1996) Tonic facilitation of glutamate release by presynaptic N -methyl-D-aspartate autoreceptors in the entorhinal cortex. Neuroscience 75: 339–344. 8. Casado M, Isope P, Ascher P (2002) Involvement of presynaptic N-methyl-D- aspartate receptors in cerebellar long-term depression. Neuron 33: 123–130. 9. Duguid IC, Smart TG (2009) Presynaptic NMDA Receptors. In: Van Dongen AM, editor. Biology of the NMDA Receptor. CRC Press; Chapter 14. 10. Sjo¨stro¨m PJ, Turrigiano GG, Nelson SB (2003) Neocortical LTD via coincident activation of presynaptic NMDA and cannabinoid receptors. Neuron 39: 641– 654. 11. Corlew R, Wang Y, Ghermazien H, Erisir A, Philpot BD (2007) Developmental switch in the contribution of presynaptic and postsynaptic NMDA receptors to long-term depression. J Neurosci 27: 9835–9845. 12. Liu SJ, Lachamp P (2006) The activation of excitatory glutamate receptors evokes a long-lasting increase in the release of GABA from cerebellar stellate cells. J Neurosci 26: 9332–9339. 13. Bidoret C, Ayon A, Barbour B, Casado M (2009) Presynaptic NR2A-containing NMDA receptors implement a high-pass filter synaptic plasticity rule. Proc Natl Acad Sci U S A 106: 14126–14131. 14. Lonchamp E, Gambino F, Dupont JL, Doussau F, Valera A, et al. (2012) Pre and Post Synaptic NMDA Effects Targeting Purkinje Cells in the Mouse Cerebellar Cortex. PLoS ONE 7(1): e30180. 15. Huang H, Bordey A (2004) Glia l glutamate transporters limit spillover activation of presynaptic NMDA receptors and influence synaptic inhibition in Purkinje neurons. J Neurosci 24: 5659–5669. 16. Shin JH, Linden DJ (2005) An NMDA receptor/nitric oxide cascade is involved in cerebellar LTD but is not localized to the parallel fiber terminal. J Neurophysiol 94: 4281–4289. 17. Glitsch MD (2008) Calcium influx through N-methyl-D-aspartate receptors triggers GABA release at interneuron-Purkinje cell synapses in rat cerebellum. Neurosci 151: 403–409. 18. Carter AG, Regehr WG (2000) Prolonged synaptic currents and glutamate spillover at the parallel fiber to stellate cell synapse. J Neurosci 20: 4423–4434. 19. Glitsch M, Llano I, Marty A (1996) Glutamate as a candidate retrograde messenger at interneurone-Purkinje cell synapses of rat cerebellum. J Physiol 497: 531–537. Presynaptic NMDARs in Cerebellar Interneurons The following drugs applied in the bath were used when appropriate: N-methy-D-aspartate (NMDA), tetrodotoxin (TTX), Mibefradil, cadmium chloride (CdCl2), zinc chloride (ZnCl2), Ifenprodil, 2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo[f]quinoxa- line-2,3-dione (NBQX), DL-2-Amino-5-phosphonopentanoic acid (AP-V), Nv-nitro-L-arginine (L-NNA), dantrolene. All drugs were purchased at Ascent. Other chemicals were purchased at Sigma. 4-Methoxy-7-nitroindolinyl-caged-L-glutamate (MNI-Glutamate) and 4-Methoxy-7-nitroindolinyl-caged-NMDA (MNI-NMDA) were kindly provided by Tocris Bioscience. Acknowledgments We thank Mariano Casado, Etienne Audinat and Philippe Ascher for valuable discussions and comments on the manuscript. Author Contributions Conceived and designed the experiments: AM IL DO TC. Performed the experiments: BR IL TC. Analyzed the data: IL BR AM TC. Contributed reagents/materials/analysis tools: DO YT. Wrote the paper: TC AM. 20. Christie JM, Jahr CE (2008) Dendritic NMDA receptors activate axonal calcium channels. Neuron 60: 298–307. 21. Pugh JR, Jahr CE (2011) NMDA receptor agonists fail to alter release from cerebellar basket cells. J Neurosci 31: 16550–5. 22. Christie JM, Chiu DN, Jahr CE (2011) Ca(2+)-dependent enhancement of release by subthreshold somatic depolarization. Nat Neurosci. 14: 62–8. 23. Christie JM, Jahr CE (2009) Selective expression of ligand-gated ion channels in L5 pyramidal cell axons. J Neurosci 29: 11441–50. 24. Bouhours B, Trigo FF, Marty A (2011) Somatic depolarization enhances GABA release in cerebellar interneurons via a calcium/protein kinase C pathway. J Neurosci 31: 5804–5815. 25. Llano I, Tan YP, Caputo C (1997) Spatial heterogeneity of intracellular Ca2+ signals in axons of basket cells from rat cerebellar slices. J Physiol 502: 509–519. 26. Trigo FF, Corrie JE, Ogden D (2009) Laser photolysis of caged compounds at 405 nm: photochemical advantages, localisation, phototoxicity and methods for calibration. J Neurosci Methods 180: 9–21. 27. Bureau I, Mulle C (1998) Potentiation of GABAergic synaptic transmission by AMPA receptors in mouse cerebellar stellate cells: changes during development. J Physiol 509: 817–831. 28. Rossi B, Maton G, Collin T (2008) Calcium-permeable presynaptic AMPA receptors in cerebellar molecular layer interneurones. J Physiol. 586: 5129–45. 29. Nowak L, Bregestovski P, Ascher P, Herbet A, Prochiantz A (1984) Magnesium gates glutamate-activated channels in mouse central neurones. Nature 307: 462– 465. 30. Llano I, Gerschenfeld HM (1993) Inhibitory synaptic currents in stellate cells of rat cerebellar slices. J Physiol 468: 177–200. 31. Mayer ML, Viklicky Jr L, Westbrook GL (1989). Modulation of excitatory amino acid receptors by group IIB metal cations in cultured mouse hippocampal neurons. J Physiol 415: 329–350. 32. Molineux ML, Fernandez FR, Mehaffey WH, Turner RW (2005) A-type and T- type currents interact to produce a novel spike latency-voltage relation-ship in cerebellar stellate cells. J Neurosci 25: 10863–10873. 33. McDonough SI, Bean BP (1998) Mibefradil inhibition of T-type calcium channels in cerebellar purkinje neurons. Mol Pharmacol 54: 1080–1087. 34. Rodrigo J, Alonso D, Fernandez AP, Serrano J, Richart A, et al. (2001) Neuronal and inducible nitric oxide synthase expression and protein nitration in rat cerebellum after oxygen and glucose deprivation. Brain Res 909: 20–45. 35. Paoletti P, Neyton J (2007) NMDA receptor subunits: function and pharmacology. Curr Opin Pharmacol 7: 39–47. 36. Trigo FF, Bouhours B, Rostaing P, Papageorgiou G, Corrie JE, et al. (2010) Presynaptic miniature GABAergic currents in developing interneurons. Neuron 66: 235–247. 37. Clark BA, Cull-Candy SG (2002) Activity-dependent recruitment of extra- synaptic NMDA receptor activation at an AMPA receptor-only synapse. J Neurosci 22: 4428–4436. 38. Duguid IC, Pankratov Y, Moss GW, Smart TG (2007) Somatodendritic release of glutamate regulates synaptic inhibition in cerebellar Purkinje cells via autocrine mGluR1 activation. J Neurosci 27: 12464–12474. 39. Larsen RS, Corlew RJ, Henson MA, Roberts AC, Mishina M, et al. (2011) NR3A-containing NMDARs promote neurotransmitter release and spike timing-dependent plasticity. Nat Neurosci 14: 338–344. PLoS ONE | www.plosone.org 13 June 2012 | Volume 7 | Issue 6 | e39983