Presynaptic NMDARs in Cerebellar Interneurons Figure 2. Wide field uncaging of MNI-glutamate reveals presynaptic NMDARs. A: MNI-glutamate was uncaged in a wide area of the recording chamber by a UV-flash through the microscope objective (see Methods) in 0.1 mM [Mg2+] and in the presence of both TTX (0.5 mM) and NBQX (5 mM). Fluorescence levels are shown before (a, left) and after (a, right) the flash. b: Reconstruction of the MLI (somatodendritic compartment in black and axon in blue). c: Ca2+ transients recorded before TTX application in response to 4 propagated action potentials in an axonal region (blue, region 5 in a) and in a dendritic region (black, region indicated «den» in a). d: MNI-glutamate uncaging in the presence of TTX and NBQX evoked a NMDAR-mediated current (left bottom trace) and Ca2+ transients in dendrites (left upper trace) as well as in axonal areas 4 and 5. B: Another experiment following the same experimental paradigm. a: Cell morphology. b: Responses to 4 propagated action potentials obtained before TTX application in axonal area 1 and in a dendritic location. c: Global uncaging of MNI-glutamate in TTX elicited Ca2+ transients both in the axonal and in the dendritic compartment (upper blue and black traces respectively), together with an inward current (bottom). doi:10.1371/journal.pone.0039983.g002 associated channels. Response onsets are also slower for axonal responses than for dendritic responses, as expected from the filtering effect of the axon cable. Nevertheless axonal responses delays are ,10 ms suggesting that axonal responses implicate NMDARs that are locate in the axon and not in neighboring cell structures. Reasons for the relative scarcity of NMDAR-mediated responses to local glutamate uncaging in axonal sites compared to the more reliable axonal responses found with wide field uncaging will be discussed below. Pharmacological Properties of Axonal NMDARs While the above experiments strongly suggest the presence of functional NMDARs in the axon they could still be interpreted as reflecting the activation of non NMDA glutamate receptors following displacement of NBQX from the receptor binding site by released glutamate. Therefore it was important to investigate whether the presumptive axonal NMDAR-mediated responses displayed features expected from NMDARs. Three characteristic features of NMDA-gated channels were investigated: Mg2+ block, block by APV, activation by NMDA. PLoS ONE | www.plosone.org 4 June 2012 | Volume 7 | Issue 6 | e39983