Recent studies have shown that acoustic over-exposure, which leads to gap detection deficits in a rodent model of tinnitus, also increases synaptic release of glutamate 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 is to investigate the presynaptic role of NMDARs in modulating synaptic release and plasticity at DCN multisensory synapses. Whole cell recordings were used to record excitatory postsynaptic currents (EPSCs) from fusiform cells in CBA mouse DCN slices. Inhibitory transmission was blocked by 10μM strychnine and 10μM gabazine. Values are reported as mean±SD. Fusiform cell LTP was induced by high frequency stimulation of parallel fibres (2) (increase of EPSC slope from 153±81pA/ms to 196±96pA/ms, **p<0.01, one-tailed ratio paired t-test, n=4). To study the effect of NMDARs on presynaptic glutamate release, miniature EPSCs (mEPSCs) were isolated with 1μM tetrodotoxin and recorded in presence of NMDAR agonist (500nM NMDA) with and without antagonist (50μM D-AP5). Friedman test with Dunn’s correction was used for statistical comparisons. In 6/12 fusiform cells, NMDA increased mEPSC frequency (decreased inter-event interval from 2257±1467ms to 1170±805ms) which was reversed by the addition of D-AP5 (inter-event interval 3340±2417ms) (control vs NMDA *p<0.05, NMDA vs NMDA+D-AP5 **p<0.01, control vs NMDA+D-AP5 p>0.99, F=11.14, n=7). Activation of NMDARs had no effect on mEPSC amplitude or decay τ (p=0.62, F=1.14 and p=0.49, F=2 respectively). Selective effect on mEPSC frequency suggests increased presynaptic release. This was further assessed in transgenic mice expressing SyGCaMP2-mCherry, a ratiometric Ca2+ sensor which allows the visualisation of Ca2+ level changes in presynaptic boutons (3). Epifluorescence imaging of DCN molecular layer showed that activating NMDARs (500nM NMDA) increases the fluorescence associated to baseline presynaptic Ca2+ levels (from 0.99±0.005 to 1.01±0.006 normalised F0, *p<0.05, Mann-Whitney test, U=0, ncontrol=4, nNMDA=5). Blocking NMDARs (50μM D-AP5) decreased the peak fluorescence response (from 1.09±0.06 to 1.06±0.05 F/F0, ***p<0.001, two-tailed paired t-test, t=5.5, n=8) evoked by parallel fibre stimulation (50pulses, 50Hz, 0.5-1mA). Multiphoton imaging of individual boutons showed that 50μM D-AP5 decreases both the number of responding presynaptic boutons (from 32% (n=130) to 21% (n=117)) and the amplitude of Ca2+ response per bouton (from 2.30±0.73 to 1.91±0.36 F/F0, ncontrol=42, nD-AP5=25, N=1). In conclusion, blocking NMDARs at DCN multisensory synapses could be used to restore basal levels of presynaptic release and in this way reverse the effect on LTP saturation and gap detection deficits observed after acoustic over-exposure (1).