Presynaptic NMDARs in Cerebellar Interneurons Figure 4. Features of NMDAR-mediated axonal responses using local uncaging. A: Responses to MNI-glutamate uncaging (arrow; AMPA receptors blocked with 5 mM NBQX; 0.5 mM TTX) at 240 and 270 mV are compared in the same axonal location in low external [Mg2+] (100 mM, upper traces) and in 2 mM Mg2+ (lower traces). B: Separate recording in 200 mM Mg2+, showing weakly voltage-dependent responses. C: Summary results. Numbers next to error bars (6 sem) indicate the numbers of cells contributing to the means. D: Addition of AP-V (50 mM; blue) abolishes the uncaging response in the presence of NBQX (5 mM). E: Uncaging experiments using the novel cage MNI-NMDA. Left: Fluorescence view of a MLI filled with Alexa 488 through the patch pipette. Middle: Current traces obtained with focused 1 ms-long laser pulses delivered in the presence of MNI- NMDA (1 mM) at various locations indicated by numbers. Right: Reconstruction of the MLI with somatodendritic compartment in black and axon in blue. doi:10.1371/journal.pone.0039983.g004 current responses both in the axonal (Fig. 5B, left) and in the dendritic domain (Fig. 5B, right). These transients rose with a time course of several hundreds of ms and they broadened during this time over a distance of ,10 mm along the axon. Importantly, axonal spots that failed to deliver a current response also failed to exhibit a Ca2+ transient. In responsive spots, the amplitude of the axonal laser pulse-elicited Ca2+ transient (mean DF/F0, 25.660.8%, n = 21 out of 6 cells) was correlated to that of the current (mean, 13.860.4 pA, n = 21 out of 6 cells; Fig. 5C, left panel). As a rule, the Ca2+ transients elicited by uncaging had a similar amplitude as those obtained using 4 propagated action potentials before TTX application (respectively DF/ F0 = 25.660.8%, n = 21 and DF/F0 = 34.761.3%, n = 15; Fig. 5C; right panel). Therefore the results indicate that a local and short activation of NMDARs in an axon spot is able to induce a large enough Ca2+ transient to increase transmitter release in the corresponding axonal area. Pharmacological Profile of NMDA Effect on Miniature Current Frequency The previous sections have examined the ability of glutamate to produce NMDAR-mediated current and Ca2+ responses in MLI axons. An alternative way to characterize the mechanism of action of NMDA is to examine the pharmacological profile of NMDA effect on miniature current frequency. In keeping with the pioneer study by Glitsch and Marty [3], we found that bath application of NMDA (30 mM) in the presence of TTX increased the frequency of mIPSCs with a frequency ratio over control of 9.9761.19 (n = 8; Fig. 6 A and D). This increase was totally prevented by the presence of MK801 (50 mM) in the bath (ratio over control: 0.8660.09; n = 4; Fig. 6D). Note that in MLIs, mEPSCs have a much lower frequency than mIPSCs, and that the two types of currents can be unambiguously distinguished on the basis of their decay kinetics [30]; therefore mEPSCs could easily be eliminated from the analysis and pharmacological block of AMPARs was not necessary in these experiments. A simple approach that can be envisaged to distinguish between direct and indirect mechanisms of NMDA action is a pharmaco- logical block of VDCCs. In the direct mechanism, NMDAR activation in the axon is likely to provide a Ca2+ rise even after total VDCC block, thus leading to mIPSC enhancement; in the indirect mechanism however, the response should be abolished by VDCC blockers. Therefore, we assessed the effects of NMDA on mIPSC frequency in the presence of 100 mM CdCl2 to block high threshold VDCCs. Under these conditions, the mIPSCs frequency ratio over control reached 7.9361.10 (n = 7) indicating that high threshold VDCCs are not required for the response (Fig. 6 B and D). The increase in mIPSCs frequency was not significantly different in Cd2+ and in control (P.0.05; unpaired t-test) although Cd2+ ions have been reported to partially block NMDARs [31]. Low threshold VDCCs are not affected by Cd2+ ions and are expressed in MLIs [32]. The T-type channel blocker mibefradil (10 mM; [33]) did not alter the potentiating effect of NMDA on mIPSCs frequency (ratio over control : 11.862.1, n = 4; P.0.05; unpaired t-test; Fig. 6D). Taken together, these results concur with those previously obtained on Purkinje neurons [3,17] to indicate that neither high threshold nor low threshold VDCCs are required for the response. They indicate that, contrary to the indirect depolarization theory, VDCC opening is not an obligatory step leading to the increase in GABA release during NMDA application. Ryanodine-sensitive Ca2+ stores have been proposed to participate in the presynaptic Ca2+ elevation triggered by preNMDARs activation [4]. In line with this proposal, addition of dantrolene (10 mM), a muscle relaxing agent acting on ryanodine receptors, significantly reduced the potentiating effect of NMDA on mIPSC frequency (ratio over control : 5.960.15, n = 4; P,0.05; unpaired t-test; Fig. 6D). An even stronger effect was obtained by combining dantrolene and Cd2+ together (ratio over control : 2.160.32, n = 4; P,0.05; unpaired t-test; Fig. 6D). These results indicate that even though VDCCs are not required for the NMDA response, they contribute together with ryanodine- sensitive Ca2+ stores to enhance the response. Such an effect could reflect a functional coupling between VDCCs and the ryanodine receptor. Note that dantrolene alone neither affects the frequency (0.3060.13 vs. 0.2760.12 Hz, n = 5; P.0.05, paired t-test; Fig. 6E, upper panel) nor the amplitude of mIPSCs (138631 vs. 112626 Hz, n = 5; P.0.05, paired t-test; Fig. 6E, lower panel). In summary, intracellular Ca2+ stores do not participate in the regulation of the resting Ca2+ concentration but that they amplify the response to NMDA. MLI axons contain neuronal NO synthase (nNOS; [34]), and MLI axon terminals have been suggested to engage an NMDAR- driven NO cascade during cerebellar LTD [16]. However NMDARs and nNOS have also been suggested to be located in PFs [8] so that local activation of NMDARs in PFs could lead to NO release and possibly to an indirect effect on MLI terminals that would be mediated by NO rather than by direct activation of NMDARs. In the presence of the nNOS antagonist L-NNA (10 mM). Under these conditions, NMDA still increased mIPSC frequency in a way that was not significantly different from that observed in control conditions (frequency ratio over control of 8.7461.25, n = 4; P.0.05; unpaired t-test; Fig. 4D). These results indicate that the NMDA-induced enhancement of GABA release in MLIs is due to the activation of NMDARs located in MLI axons rather than to an indirect mechanism involving NO release from neighboring structures. The effects of NMDA on synaptic activity were readily observed in normal TTX- containing BBS. This indicates that the preNMDARs activation occurs even in the presence of the normal Mg2+ concentration (1 mM) without any pre-depolarization to relieve the Mg2+ block. To explain this result, it has been postulated that preNMDARs could incorporate NR2C and D subunits in their structure which would render them less sensitive to Mg2+ inhibition than their NR1 and NR2A/B counterparts [3,4]. Alternatively, axonal depolarization could amplify pre- NMDAR activation. Zn2+ ions are widely used to distinguish NR2A subunits as they appear to be far more selective for NR1/ NR2A receptors than for any other NR2 subunit-containing PLoS ONE | www.plosone.org 7 June 2012 | Volume 7 | Issue 6 | e39983