Presynaptic NMDARs in Cerebellar Interneurons Figure 7. NMDA application increases premini frequency in voltage-clamped MLIs. A: Spontaneous synaptic currents in the presence of TTX (preminis are identified as events having a peak amplitude of ,30 pA; there is 1 such event in the upper trace, plus one somatodendritic miniature) and after further bath application of 20 mM NMDA (lower trace: 5 preminis plus 2 minis). The soma is voltage clamped at 260 mV. 2 of the miniature events, one in control and one in NMDA, have their peaks clipped off. B: Plots of 20–80% risetime as a function of peak amplitude in control (2 min duration) and in NMDA (also 2 min), from the same experiment as in A. Conventional miniature currents (minis) appear as a cluster of events with peak amplitudes .30 pA and risetimes ,0.7 ms, and presynaptic miniature currents (preminis) appear as a non overlapping cluster of events with peak amplitudes ,30 pA and risetimes .0.5 ms. Note that the frequencies of both preminis and minis increase in response to NMDA application. C: Peak amplitude histograms from the data in B (gray: control; open bars: NMDA). D: Dots: summary of 8 experiments (5 with 20 mM NMDA, and 3 with 50 mM NMDA), showing ratios of both premini and mini frequencies in NMDA over control periods. Only 1/8 experiment fails to show an increase in the premini frequency in response to NMDA. Open squares: summary of 4 experiments performed in the presence of NBQX (20 mM), Cd2+ (100 mM) and NMDA (50 mM). Experiments have been carried out in normal extracellular [Mg2+] (1 mM). doi:10.1371/journal.pone.0039983.g007 from depolarizing the axon compartment does not abolish the NMDA response. Uncaging Experiments The results obtained with wide field uncaging confirm that most axons are responsive to NMDA, but they also indicate that this sensitivity is restricted to a small number of varicosities. In addition, they rule out Ca2+ diffusion from the somatodendritic compartment as an explanation for NMDA-induced axonal Ca2+ elevations under voltage clamp because no Ca2+ gradient from soma/dendrite to axon terminals was observed. Local uncaging either of NMDA or of glutamate in conditions where both AMPA-selective and kainate-selective glutamate receptors are blocked, leads to the generation of an axonal current that can be collected in whole-cell recording. These experiments allow for a much more precise and reproducible agonist application than the previous approach based on iontophoresis [20,21], but they nevertheless suffered from some technical limitations. First, axonal structures were not always available, since the axon was sometimes located under the recording pipette. In other cases the axon was visible but plundged into the slice tissue. Because the loss of 405 nm laser intensity due to scattering is approximately two-fold per 18 mm [26], the uncaging efficiency of local laser stimulation declines quickly with depth. This explains why responsive axonal stretches were immediately near the slice surface. Second, experience proved that only a limited number of laser flashes could be applied before photodamage was evident, so that a relatively small number of locations (typically not more than 5) were tested in a given experiment. These limitations explain that only a minority of uncaging experiments revealed any axonal sensitivity to NMDAR, without implying the existence of non responsive axons. In responding axons, only 28% of the probed locations gave rise to a detectable response. This result is in keeping with the fact that in cultured MLIs, 39% of the patches excised from axon terminals did not display functional NMDARs [5] indicating that a signif- icant proportion of varicosities does not contain NMDARs. The sparse nature of axonal NMDA signaling may account in part for the previous failure to detect axonal NMDARs using iontophoretic NMDA applications [20,21,37] as well a two-photon uncaging of MNI-glutamate [21]. The exact nature of the responsive spots remains to be determined but they presumably include basket terminals onto Purkinje cells since these structures display presynaptic NMDAR immunostaining [38] and since they appeared especially responsive to NMDA in the present work. PreNMDAR Activation Leads to Ca2+ Transients In the presence of OGB-1 in the intracellular recording solution, we were able to simultaneously record Ca2+ transients and NMDA-evoked currents. The amplitudes of the transients obtained with 1 ms laser pulses were comparable to those of summed transients evoked by four action potentials in close succession. It seems therefore plausible that a short-lived activation of preNMDARs leads to a robust release of GABA. These transients were found to spread along the axon over a distance of ,10 mm, possibly reflecting diffusion as well as the recruitment of ryanodine-sensitive intracellular Ca2+ stores [4]. Local amplifica- tion by intracellular Ca2+ stores may also account for the negative results obtained with aspartate iontophoresis by [20], since the constant use of the SERCA pump inhibitor cyclopiazonic acid in these experiments may have emptied Ca2+ stores. Taken together, our data indicate that this axonal sensitivity to NMDA is strong enough to fully account for the effects observed on miniature currents. Pharmacological Profile of the mIPSC Response to NMDA According to the indirect mechanism the mIPSC frequency increase elicited by NMDA should be highly sensitive to VDCC blockers. However we found that neither the general VDCC blocker Cd2+, or the T-type VDCC blocker mibefradil, were able to inhibit this effect significantly. These results together with those of a previous study [17], demonstrate that activation of VDCC is dispensable for the mIPSC response. The strength of the inhibitions observed with NR2 subunit antagonists (i.e. Zn2+ and ifenprodil) indicate that the NR3A subunit is not present presynaptically unlike in the neocortex where NR3A-containing preNMDAR promote neurotransmitter release and spike timing- dependent plasticity [39]. Indeed, the expression of NR3A in cerebellum has been shown to be barely detectable after 10 days PN [40]. The half-decay time of preNMDARs current is consistent with mixed involvement of NR2A and -B subunits since it matches the deactivation kinetics of the currents recorded in HEK cells expressing recombinant NMDARs containing either NR2A or NR2B subunit [13]. Functional Role of preNMDARs In MLIs, activation of preNMDARs i) increases spontaneous and miniature IPSC frequency in Purkinje cells and MLIs; ii) triggers the onset of depolarization-induced potentiation of inhibition (DPI) in immature Purkinje cells [4] and iii) leads to I- LTP in MLIs [12]. Application of D-APV has been shown to slightly decrease the frequency of mIPSCs recorded in Purkinje cells [3,15] suggesting a tonic activation of NMDARs. These results suggest that the regulation of IPSC frequency occurs as soon as the ambient glutamate concentration exceeds normal levels, whether as a result of some local unbalance or because of strong or sustained parallel fibre activity; because of the NMDAR- mediated increase in spontaneous inhibition on postsynaptic MLIs and Purkinje cells, the effects of ambient glutamate on the mean level of cell firing in the molecular layer are then kept within acceptable limits. On a somewhat longer time scale (minutes) the onset of DPI triggers a regulatory loop by which increased MLI inhibition corrects excessive firing of Purkinje cells. Finally I-LTP fulfills a similar role on a still longer time scale (hours). Therefore PLoS ONE | www.plosone.org 11 June 2012 | Volume 7 | Issue 6 | e39983