Recent studies have shown that acoustic over-exposure, leading to gap-detection deficits in a rodent model of tinnitus, also increases glutamate release at dorsal cochlear nucleus (DCN) multisensory synapses (1). This activates NMDA receptors (NMDARs) and leads to saturation of long-term potentiation (LTP) (1). The aim of this study was to investigate the role of NMDARs in modulating Ca2+-dependent presynaptic activity and glutamate release in the DCN molecular layer. Whole-cell recordings were used to record miniature excitatory postsynaptic currents (mEPSCs) from DCN fusiform cells in CBA mouse slices. Miniature EPSCs were isolated with tetrodotoxin (1μM) and recorded in the presence of NMDAR agonist (500nM NMDA) with and without antagonist (50μM D-AP5). All values are reported as mean±SD. Unless otherwise stated, Friedman test with Dunn’s correction was used for statistical comparisons. In 8 out of 12 fusiform cells, NMDA increased mEPSC frequency (decreasing inter-event intervals from 0.68±0.41s to 0.42±0.31s, p<0.05) which was reversed by the addition of D-AP5 (inter-event interval 1.0±0.89s, NMDA vs NMDA+D-AP5 p0.99, F=7, n=8). NMDA did not affect mEPSC amplitude (p=0.2, F=1.85, RM one-way ANOVA with Holm-Sidak’s correction, n=8) or decay time constant (p=0.24, F=3.3, n=8). Selective effect on mEPSC frequency indicates a presynaptic effect of NMDARs. This was further assessed in transgenic mice expressing SyGCaMP2-mCherry, a ratiometric Ca2+ sensor reporting Ca2+ level changes in presynaptic boutons (2). Epifluorescence imaging of molecular layer showed that NMDA increased baseline fluorescence compared to control slices (pinteraction<0.001, F=3.3, mixed ANOVA, control n=5, NMDA n=7) after 12 min of perfusion (p<0.05, F=9.9, univariate ANOVA). Fluorescence responses evoked by parallel fibre stimulations were recorded in absence and presence of D-AP5. D-AP5 decreased the peak fluorescence compared to control slices (from 101.2±5.9% to 77.1±8.6% F/F0, p(interaction)<0.01, F=2.4, mixed ANOVA, control n=7, D-AP5 n=8) after 25 min of perfusion (p<0.001, univariate ANOVA). Multiphoton imaging showed that D-AP5 decreased the peak F/F0 per bouton (from 1.4±0.2 to 1.3±0.2 F/F0, p<0.0001, Wilcoxon test, W=-1281, n=57 boutons, N=4 slices) without changing the number of responding boutons (from 62.9±15.9% to 52.3±22.2%, p=0.06, unpaired t-test with Welch’s correction, t=1.9, n=24, N=4). In conclusion, activation and inhibition of NMDARs lead to an increase and a decrease of presynaptic calcium respectively, at DCN multisensory synapses. This study suggests that the increase of glutamate release observed after acoustic over exposure (1) is likely to be mediated by a presynaptic action of NMDA at those synapses.