In the present work we show that NMDA superfusion leads to axonal Ca2+ elevations under somatic voltage clamp and we map the NMDA sensitivity over the entire cell using wide field and local uncaging of NMDAR agonists. These methods reveal discrete spots of high sensitivity in the axon domain indicating that MLI could contain functional preNMDARs. Finally, we reexamine the pharmacological properties of NMDA-induced mIPSC enhance- ment, finding little sensitivity to VDCC blockers. Altogether the data provide a characterization of the effects of preNMDARs activation on cellular current and Ca2+ signaling. Results Activation of Axonal NMDARs Under Somatic Voltage Clamp Conditions The original suggestion that MLIs have functional pre- NMDARs has emerged from the observation that bath application of NMDA strongly increases the frequency of mIPSCs recorded in the two cell types that are innervated by MLIs i.e. Purkinje cells and other MLIs [3]. It was however immediately pointed out that, as an alternative or complementary interpretation to the direct activation of preNMDARs, axonal depolarization could result from activation of somatodendritic NMDAR following passive spread of somatic depolarization along the axon cable [3]. The latter component of the response would lead indirectly to GABA release, as the secondary depolarization of synaptic terminals would locally activate VDCCs producing an elevation of GABA release. Such a mechanism was recently described in the case of subthreshold somatic depolarisations [22,24]. In fact, Christie and Jahr (2008, [20]) proposed that the NMDA-evoked depolarization exclusively arises from this indirect mechanism, and does not involve a direct action of NMDA on the axon. In this view, all NMDARs in MLIs would be somatodendritic. According to this hypothesis, somatic depolarization is an obligatory intermediate between activation of somatodendritic NMDARs and the sub- sequent increase in axonal Ca2+ concentration. If this is the case, then clamping the soma at a negative holding potential should prevent the effect entirely [20]. We therefore examined local axonal Ca2+ signalling following bath application of NMDA by performing 2-photon imaging of axon stretches in voltage clamped MLIs. In each experiment the identification of the recorded neurite as the axon was confirmed by analysis of action potential induced Ca2+ transients, which are markedly larger in the axon than in dendrites [25]. Following this identification, TTX (0.2 mM) and NMDA (50 mM) were sequentially added to the bath. In 7 MLIs, NMDA elicited inward currents with an average peak value of 2668 pA under somatic voltage clamp (holding potential, 260 mV). Parallel to these currents, the Ca2+-de- pendent fluorescence in axonal varicosities increased in all experiments, with DF/F0 peak values ranging from 12 to 177% (63610%, 19 varicosities from 7 cells). The fluorescence changes were transient, and returned to baseline occurring within 2 to 4 minutes after washing the agonist. The two largest Ca2+ transients in this series of experiments were obtained in basket cell terminals onto Purkinje cell somata. Fig. 1 illustrates one of these experiments, in which a basket cell was challenged with NMDA while imaging boutons on the Purkinje cell layer (Fig. 1A–C). The NMDA-induced current was accompanied by robust Ca2+ transients in the boutons directly in contact with the Purkinje cell soma. These data indicate that NMDA application is able to elicit large Ca2+ transients in basket cell terminals in spite of somatic voltage clamp. In 5 additional experiments, NMDA applications were performed in the presence of 6 mM external Ca2+. The Ca2+- Presynaptic NMDARs in Cerebellar Interneurons dependent fluorescence changes were larger with a peak DF/F0 amplitude of 340689% (n = 5 cells). The effect had a gradual onset: even though a Ca2+ increase was observed as soon as NMDA was applied, the response typically grew over a time course of several minutes in the continuous presence of NMDA, and it declined rather slowly after washing out the NMDA, with a time for returning to baseline of 2 to 5 min after agonist removal. Such slow kinetics are consistent with a participation of in- tracellular Ca2+ stores in the NMDA-induced Ca2+ response. In summary, bath application of NMDA elicits significant axonal responses under somatic voltage clamp, both with 2 mM and with 6 mM extracellular Ca2+. The axonal Ca2+ response cannot be attributed to voltage escape following activation of somato-dendritic NMDARs since the somatically recorded NMDA-induced currents in these experiments did not exceed 50 pA. Thus, somatic voltage clamp must have prevented depolarization spread from the dendrites to the axon. The axonal Ca2+ rise cannot be attributed to Ca2+ diffusion from the somatodendritic compartment since instead of the continuous concentration gradient expected from a diffusion process, a patchy response pattern was observed, with high response locations (which were often found in basket cell terminals) following much lower response locations along the axon course. Therefore these results, like those of similar experiments previously reported on stellate cells [15] are potentially difficult to reconcile with the indirect hypothesis. Wide Field Glutamate Photorelease The preceding experiments leave the possibility open that NMDA would not bind directly to the MLI axon displaying the Ca2+ response, but to some other structure (not necessarily in the same cell), which would lead to the axonal response by unknown intermediary steps. Bath NMDA applications are not appropriate in this context since they lead to protracted activation of NMDARs with an imprecise timing. By contrast uncaging experiments provide a much more precise control of NMDAR activation timing. In the next series of experiments, we produced step increases in glutamate concentration by adding the photo- sensitive precursor of glutamate MNI-glutamate and by applying UV flashes to the entire preparation. Slices were incubated in a low Mg2+ (0.1 mM) HEPES-buffered saline supplemented with MNI- glutamate (final concentration, 0.9 mM) for at least 0.5 h before the beginning of the experiment. MLIs were perfused with Oregon Green BAPTA 1 (OGB-1) through the patch pipette and kept in the presence of NBQX (5 mM) to block AMPARs. Alexa 488 was added to resolve long axonal stretches. Ca2+ imaging was performed using a CCD camera (see methods). As depicted in Fig. 2Aa (left panel) the plane of focus typically contained several regions of interest that were selected according to their presumptive axonal or dendritic nature. A train of 4 propagated action potentials led to larger Ca2+ transients in the axonal compartment and therefore confirmed the identification of axon and dendrites (compare blue and black traces in Fig. 2Ac and Fig. 2Bb). After application of TTX, a UV flash yielded somatic currents (3664 pA, n = 23; Fig. 2Ad and Fig. 2Bc) accompanied by Ca2+ transients in dendrites (black traces in Fig. 2Ad and Fig. 2Bc; DF/ F0:33.765.0%, n = 11) as well as in certain regions of the axon (Fig. 2Ad, blue traces 4 and 5; Fig. 2Bc, blue trace 1; DF/ F0:11.261.7%, n = 13). Responsive axonal varicosities were found in 5 out of 6 cells. As discussed above, in the context of the experiments with bath application of NMDA, since the soma was under voltage clamp the only mechanism that could account for a participation of somatodendritic NMDARs to any axonal PLoS ONE | www.plosone.org 2 June 2012 | Volume 7 | Issue 6 | e39983