Presynaptic NMDARs in Cerebellar Interneurons Figure 1. Presynaptic Ca2+ elevation in response to bath application of NMDA in a voltage clamped basket cell. The cell was voltage clamped at a holding potential of 260 mV in normal extracellular [Mg2+] (1 mM). It was perfused with the Ca2+ dye OGB-5N and part of its axon was imaged using 2-photon Ca2+ imaging. NMDA was added to the bath perfusion during the time indicated by bars (0.5 mM TTX throughout), while imaging a basket terminal belonging to the recorded cell. A: Fluorescence images from 2-photon laser scanning Ca2+ imaging at rest (left) and during the peak of the NMDA-induced current (right). B: Superposition of transmitted light and fluorescence images, showing that the fluorescent structure illustrated in A is a basket cell terminal in contact with a Purkinje cell soma. C: Time course of the NMDA-induced current and of the calcium- dependent fluorescent changes in 2 ROIs of the terminal as indicated by boxes in A, with matching colors in traces and in boxes. doi:10.1371/journal.pone.0039983.g001 response would be Ca2+ diffusion along the axon. This can however be ruled out since responsive distal axonal spots follow irresponsive proximal spots as illustrated in Fig. 2Ad. In the case of the diffusion hypothesis, the Ca2+ concentration would be expected to gradually decrease from the soma. Importantly, axonal responses rose as fast as dendritic ones, with a delay shorter than the time resolution of imaging (50 ms). This argues against the possibility of slow intermediate steps intervening between NMDAR activation and axonal Ca2+ elevation. Local Glutamate Photorelease in the Axon To further characterize the local sensitivity of MLI axons to NMDA, we restricted photorelease of glutamate by using focused laser illumination [26]. In these experiments, like before, action potentials were blocked by TTX, and the local sensitivity of MLI neurites to glutamate was examined before and after blockage of AMPA receptors, using short and sharply focused laser pulses (405 nm; 0.1 to 1 ms). As shown in Fig. 3Ac, the uncaging spot had a half width of ,1 mm. Releasing glutamate in the absence of AMPA receptor blocker elicited somatic current transients that differed markedly according to the location of the laser spot (Fig. 3A,B). Previous work suggest that AMPA receptors are present in both somatodendritic and axonal compartments of MLIs [27,28], so that these experiments were expected to activate a mix of AMPA and NMDA receptors in both cases. When applied to the somato-dendritic compartment, laser pulses evoked robust current responses with rather high amplitude (139619 pA, n = 12 locations out of 12 cells; Fig. 3A and C) and fast kinetics (20/80% rise time: 1.060.1 ms, n = 12; half decay time: 3064 ms, n = 12; Fig. 3C). Axonal responses obtained under the same conditions displayed a smaller amplitude (2866 pA, n = 67 varicosities out of 16 cells; Fig. 3A and C) and slower kinetics (20/80% rise time: 2.660.5 ms, n = 9 responses from 9 cells; half decay time: 84616 ms, n = 9 responses from 9 cells; Fig. 3C). The somatodendritic responses were often biphasic and displayed a fast component with a time constant of decay smaller than 5 ms (Fig. 3Ab, black trace), presumably mediated by AMPARs. To isolate the contribution of NMDARs, the same experiments were performed in the presence of NBQX (5 mM). Somatodendritic responses were then found in every cell, however axonal responses were detected in only 12% of the cells (24 out of 202 cells). In responsive cells laser pulses applied along the axons gave rise to detectable currents in 28% of the locations. Uncaging of MNI- glutamate on dendritic spots gave rise to currents with an average amplitude of 4264.2 pA (n = 26 locations out of 14 cells) with a 20/80% rise time of 8.362.8 ms (n = 19 out of 8 cells) and a half decay time of 141618 ms (n = 20 out of 9 cells). The same experiments performed in the axon elicited currents displaying an average amplitude of 1361.2 pA (n = 90 varicosities out of 24 cells), a 20/80% rise time of 1761.4 ms (n = 33 responses out of 7 cells) and a half decay time of 17268 ms (n = 33 responses out of 7 cells). Axon identification is unambiguous in MLIs; however it could be envisaged that the responses that were attributed to axonal NMDARs were in fact elicited in dendritic structures that happened to be close enough to the targeted axon to be activated by glutamate diffusing away from the release spot. To address this possibility, we assessed the spatial resolution achieved by laser spot uncaging of MNI-glutamate with 1 ms pulse duration in the presence of NBQX (Fig. 3Bd). We found a very abrupt drop in sensitivity when moving the laser spot location away from the neurite, with a length constant of 1.4 mm. In view of these controls sensitive axonal spots that were located within 5 mm from any potential somatodendritic structure were rejected from the above analysis. These experiments demonstrate that sizable current responses can be elicited from both axonal and dendritic locations following local and short activation of either AMPARs or NMDARs. The results reveal quantitative differences depending on neurite type and pharmacological conditions. Responses are larger and faster for AMPARs than for NMDARs, in accordance with the slower opening kinetics of NMDAR-associated channels than AMPAR- PLoS ONE | www.plosone.org 3 June 2012 | Volume 7 | Issue 6 | e39983