all three effects may be seen as contributing to reestablishing near- normal conditions in case of excessive neuronal activity. The strong inhibition of miniature current frequency observed with a combination of VDCC and ryanodine receptor antagonists suggest that the activation of preNMDARs triggers a signaling module involving both VDCCs and Ca2+ stores in the amplifica- tion of the initial rise in presynaptic Ca2+. Moreover, the dectection of nNOS [34] and PSD-95 [41] immunoreactivity in MLI terminals led to propose the existence of NMDAR-contain- ing signaling complexes [16]. Such units are likely to contribute to the NO-dependent form of LTP that is produced at the PF-stellate cell synapse by pairing low frequency stimulation of PFs with postsynaptic depolarization [42]. An NMDA/nNOS unit in MLI was also suggested to support the induction of LTD at the PF-PC synapse [16] although a similar cascade has been shown to directly take place in PFs [13]. The recruitment of the NMDAR-NOS Ca2+ signaling pathway in MLIs could simultaneous trigger LTD of PF-Purkinje cell synapses and LTP of PF-MLI synapses, a coincidence that may result in a strong attenuation of Purkinje cell activity. Materials and Methods Ethics Statement Sprague–Dawley rats were purchased at Elevage Janvier (Saint Berthevin, France). They were housed at the animal house of Centre Universitaire des Saints Pe`res, which is approved by the ‘Pre´fecture de Police’ following inspection by Veterinary Services of the city of Paris, and representatives of the French Ministry of Research and the Ministry for Health, in agreement with the European Directive 86/609/EEC regarding the protection of animals used for experimental purposes (approval number A- 750607). Slice Preparation Postnatal day 12–16 rats were killed by decapitation under general anaesthesia following inhalation of the volatile anaesthetic isourane. Parasagittal (200 mm) cerebellar slices were cut with a vibroslicer (Leica VT1200S, Germany) in ice-cold bicarbonate buffered saline (BBS) solution containing the following (in mM): 125 NaCl, 2.5 KCl, 2 CaCl2, 1 MgCl2, 1.25 NaH2PO4, 26 NaHCO3, and 10 glucose (saturated with 95% O2 - 5% CO2), pH 7.3. The slices were incubated at ,34uC for 30 min and were then stored at room temperature. During recordings, the slices were superfused with the above solution (1–1.5 ml/min) at room temperature (20–23uC) except for uncaging experiments. Stellate and Basket Cells MLIs were identified using an upright microscope (Axioskop, Zeiss, Germany) with differential interference contrast (DIC) optics, a 60X Olympus objective, 0.90 water immersion objective, and a 0.63 condenser. In the present report, the same results were found in all interneurons tested irrespective of their location in the molecular layer, we therefore refer collectively to stellate and basket cells as MLIs. Recording and Analysis of IPSCs MLIs were maintained under voltage clamp in the whole-cell recording (WCR) configuration at a holding potential of 270 mV. The intracellular solution contained (in mM) 150 KCl, 4.6 MgCl2, 0.1 CaCl2, 10 HEPES-K, 1 mM EGTA-K, 0.4 Na-GTP, and 4 Na-ATP, pH 7.3. Tight-seal WCRs were obtained with borosilicate pipettes (4–6 MV) from superficial somata using an EPC-9 amplifier (HEKA Electronik, Darmstadt, Germany). Series Presynaptic NMDARs in Cerebellar Interneurons resistance values ranged from 15 to 25 MV and were compensated for by 60%. Currents were filtered at 1.3 kHz and sampled at a rate of 250 msec/point. N-methyl D-aspartate (NMDA) was added to the bath at 30–50 mM. mIPSCs and ‘‘preminis’’ were recorded in the presence of 0.5 mM TTX. For pharmacological studies, synaptic current parameters were compared during a control period preceding NMDA application and a test period lasting from 2 to 3 min after onset of the application. Detection and analysis of IPSCs were performed off-line with routines written in the IGOR Pro programming environment (Wave- Metrics). Note that in MLIs, most spontaneous synaptic currents were blocked by bicuculline (20 to 50 mM) and were therefore mediated by activation of GABAA receptors. IPSCs and EPSCs could be distinguished based on the decay kinetics of the two currents which differ by an order of magnitude as reported [30]. For specific experiments, Zn2+ was buffered with tricine [N- tris(hydroxymethyl)methylglycine], as described [43]. Wide Field Uncaging of MNI-glutamate Whole field photolysis of MNI-glutamate was achieved using a pulsed xenon arc lamp (Till Photonics, Germany) as described [44]. Briefly, a high intensity (0.5 ms duration; 80 J) discharge of UV light (36067.5 nm) was reflected onto the plane of focus using a dichroic mirror and the 60X water immersion objective. For uncaging experiments, the perfusion was turned off to minimize consumption of the cages. A HEPES-buffered solution supple- mented with NaHCO3 to control internal pH was used (composition in mM: 135 NaCl, 4 KCl, 2 NaHCO3, 25 glucose, 2 CaCl2, 0.1 MgCl2 and 10 HEPES, pH 7.4 with NaOH). Local Uncaging of MNI-glutamate and MNI-NMDA Laser photolysis was carried out by implementing a 405 nm diode laser (Point Source Iflex 2000) coupled with a single mode optical fibre into the epifluorescence condenser of a commercial microscope (Zeiss Axioskop FS1) as described previously [26]. In this implementation the fibre output of the laser was expanded with a 40 mm focal length positive lens in a dual LED lamphouse (OptoLED, Cairn Research, UK) and reflected with a 45u dichroic mirror (425 DCXR, Chroma) into the epifluorescence condenser of the microscope. The epifluorescence condenser was previously adapted to accommodate output of a TILL monochromator (Polychrome 2, TILL Photonics, Germany) and the final dichroic reflector changed to an extended UV type (T490LP, Chroma). After initial alignment, the laser fibre and first lens were adjusted on three axes to position and focus the uncaging spot in the microscope focus. A spot of approximately 1 mm diameter was formed at the focal plane in a 100 mM pyranine solution using a CCD camera (TILL Photonics, Germany). The straight-through path of the LED lamphouse received the fibre output of the monochromator for epifluorescence excitation at 488 nm (slit with 10 nm). Calcium Imaging Two different setups were used for Ca2+ imaging experiments, either a custom two-photon laser-scanning microscopy (2PLSM) system ([45] and references therein) or a single-photon system. The 2PLSM setup was used to assess the axonal Ca2+ rise induced by bath application of NMDA. Other experiments were performed on a single photon setup from TILL Photonics (Germany) based on a Polychrome 2 monochromator and a Peltier-cooled CCD camera (IMAGO QE; 137661040 pixels; pixel size: 244 nm after 53X magnification and 262 binning). Excitation was at 480 nm and images were acquired with emission at 510–550 nm. To induce axonal [Ca2+]i rises, trains of 4 action PLoS ONE | www.plosone.org 12 June 2012 | Volume 7 | Issue 6 | e39983