Presynaptic NMDARs in Cerebellar Interneurons Figure 3. Activation of presynaptic ionotropic glutamate receptors using local glutamate uncaging. A: Responses obtained in TTX in the absence of NBQX. a: Fluorescence image of a MLI filled with 40 mM Alexa 488 via the patch pipette. b: Current responses to 0.3 ms-long laser ashes that locally delivered glutamate from its photolabile precursor MNI-glutamate (1 mM) in the soma (1, black trace) and at various axonal locations (blue). c: The size of the actual uncaging spot was measured by drawing a line accross its image in fluorescence and fitting the projection of the spot on the line by a Gaussian curve (AU: fluorescence arbitrary units; full width at half maximum = 1.38 mm). B: A similar experiment performed in the presence of NBQX (5 mM). a: Fluorescence image. b: Current responses to 1 ms-long laser pulses at various locations indicated by numbers on the cell reconstruction. c: Spatial resolution of axonal uncaging using MNI-glutamate in the presence of NBQX. To construct this curve the relative location of the laser and of the preparation was moved in the direction orthogonal to the neurite, in 4 separate experiments. The data were fit to an exponential decay with a space constant of 1.40 mm. C: Average amplitude (left), 20–80% risetime (middle) and half-decay time (right) of the currents evoked in dendrites (den) and axons (ax) by local uncaging of MNI-glutamate in TTX, in the absence (control) or in the presence (NBQX) of NBQX (5 mM). Error bars indicate 6 sem; associated numbers indicate numbers of current traces contributing to the mean. All these experiments were performed in low extracellular [Mg2+] (0.1 mM). doi:10.1371/journal.pone.0039983.g003 Because of the well known blocking action of external Mg2+ ions on NMDA-sensitive channels [29] we performed experiments in a low external Mg2+ saline ([Mg2+] ,0.1 mM). Under these conditions, the currents observed in response to axonal MNI- glutamate uncaging in the presence of NBQX were weakly dependent on the holding voltage (Fig. 4Aa,C). However, when Mg2+ was added to the bath, the currents became clearly voltage dependent, and started to display the characteristic outward rectification of NMDAR-coupled channels (Fig. 4Ab,C). Interme- diate voltage dependence was observed for 200 mM Mg2+ in the bath (Fig. 4B,C). Direct identification of the responses as arising from NMDARs activation was provided by the fact that when the specific NMDAR antagonist AP-V (50 mM) was added, the uncaging response was blocked (n = 3; Fig. 4D). Finally we investigated the sensitivity of the axonal channels to NMDA by using a novel NMDA cage instead of MNI-glutamate. Fig. 4E shows current traces obtained by local uncaging of MNI-NMDA (1 mM) in response to 1 ms laser pulses at various locations as indicated. Axonal current responses displaying an average amplitude of 11.563.5 pA were recorded in 6 cells out of a total of 29. Collectively, the results indicate that the axonal responses obtained by local MNI-glutamate uncaging in the presence of NBQX reflect the activation of NMDARs. Axonal Ca2+ Responses to Local NMDAR Activation Next we tested whether local axonal glutamate uncaging (Fig. 5A, blue arrowhead) could produce Ca2+ transients that would be associated with the current responses. Local MNI- glutamate uncaging in TTX gave rise to Ca2+ transients and to PLoS ONE | www.plosone.org 5 June 2012 | Volume 7 | Issue 6 | e39983