Figure 6. NMDAR activation increases the mIPSC frequency in MLIs. A: mIPSCs recorded from a representative MLI under control conditions (0.5 mM TTX, left) and in the presence of 30 mM NMDA (right). A modest inward current shift (control level indicated by continuous line on the right) represents activation of somatodendritic NMDARs. Note the marked mIPSC frequency increase. B, C: Similar experiments were performed in the continuous presence of Cd2+ (100 mM; B) or ifenprodil (10 mM, Ifen, C). D: Summary data. The percentage of increase of mIPSC frequency obtained with NMDA is plotted in various conditions: no drug (NMDA), MK801 (50 mM), Cd (Cd2+, 100 mM), Mibe (mibefradil, 10 mM), Da (dantrolene, 10 mM), Cd+Da (Cd2+ and dantrolene), L-NNA ((L)N-nitroarginine, 10 mM), Ifen (ifenprodil, 10 mM) and Zn (Zn2+, 300 nM). E: The presence of dantrolene (10 mM) does not significantly affect the frequency or the amplitude of mIPSCs. All experiments were carried out in normal extracellular [Mg2+] (1 mM). doi:10.1371/journal.pone.0039983.g006 mention that Zn2+, ifenprodil, mibefradil, L-NNA do not alter mIPSC frequency by themselves. NMDA Application Increases the Frequency of Preminis The results of Fig. 1 show that under somatic voltage clamp conditions, where the indirect mechanism of NMDA-induced GABA release is blocked, activation of axonal NMDARs leads to a large elevation of the cytosolic Ca2+ concentration. To test whether this effect was able to increase neurotransmitter release, we examined a new class of miniature currents referred to as « preminis » [36]. Preminis reflect the activation of presynaptic GABAARs through individual vesicular release events and therefore require neurotransmitter release from the recorded cell. Presynaptic NMDARs in Cerebellar Interneurons They exhibit specific amplitude and kinetics that set them apart from conventional miniature events. A representative experiment is illustrated in Fig. 7. Under control conditions (TTX, 0.5 mM) preminis had a peak amplitude smaller than 30 pA and risetime values ranging from 0.5 to 6 ms (Fig. 7A, B). By contrast conventional miniature events had amplitudes of 30–300 pA and homogeneous risetime values of about 0.4 ms (Fig. 7A, B). In the presence of NMDA, the recording noise increased, but it was still possible to identify the two separate classes of events, and the frequencies of both preminis and conventional miniatures increased (respectively from 0.43 to 0.93 Hz, and from 0.15 to 0.84 Hz; Fig. 7 A–C). Summary results from 7 cells gave a mean increase of 2.5-fold for preminis, and of 4.1-fold for conventional miniature currents (Fig. 7D). Like the previous results of Fig. 6, the premini frequency increase must be due to activation of preNMDARs because the soma was under voltage clamp. The results of Fig. 7 indicate that the elevation of the axonal Ca2+ concentration elicited by preNMDAR activation is able to induce transmitter release in spite of the somatic voltage clamp. To confirm that premini activation was not due to an indirect mechanism involving neighboring MLIs, mIPSCs and preminis were recorded in the presence of NBQX (20 mM) and Cd2+ (100 mM) to block the indirect pathway. In the presence of NMDA, the frequency of preminis increased from 0.12 to 0.46 Hz. Averaged results from 4 cells gave a mean increase of 3.3-fold for preminis and 5.2 for minis (Fig. 7D; open square symbols). These values are not significantly different from these obtained without blockers, thus confirming that the increase in premini frequency is due to activation of axonal NMDARs in the recorded cell. It is worth noting that the augmentation of premini frequency is of the same order of magnitude as that of conventional miniatures (which originate from unclamped MLIs). The increase measured for preminis is actually smaller than that of conventional miniatures. However, the first value is an underestimate because NMDA decreases the length constant of the axon cable by increasing the membrane permeability of this cable, thus de- creasing the effective surface area from which preminis are collected. Since this effect could easily explain the difference between the 2.5-fold increase in premini rate and the 4.1-fold increase in mini frequency, the results suggest that the entire scope of the NMDA-induced frequency increase can be accounted for by the direct activation of axonal NMDARs. Discussion Altogether our results fail to substantiate an indirect mechanism for the responsiveness of MLI axons to NMDA, and suggest instead a direct mechanism reflecting the activation of NMDARs in the axonal domain of these cells. Here we briefly review the key new findings that favor the direct mechanism, and we proceed with a discussion of the specific features of axonal NMDAR- mediated signaling that emerge from the present work. NMDA-induced Axonal Ca2+ Rise and Subsequent GABA Release under Somatic Voltage Clamp Clamping the somatic potential at a negative value did not prevent NMDA-induced axonal responses. Large axonal Ca2+ elevations were still elicited by NMDA application and further- more, these Ca2+ elevations were able to elicit GABA release as demonstrated by the fact that the rate of preminis was significantly enhanced. Thus, contrary to the predictions of the indirect mechanism hypothesis, preventing somatodendritic NMDARs PLoS ONE | www.plosone.org 9 June 2012 | Volume 7 | Issue 6 | e39983