Nitric oxide (NO) signalling is implicated in several neurodegenerative diseases through induction of high NO release. However, its exact contribution to degeneration remains elusive due to the complexity of downstream nitrergic targets. High levels of NO can induce post-translational modifications which are associated with neuronal degeneration (Steinert et al., 2010; Nakamura et al., 2015). NO reacts with superoxide anions to form cytotoxic peroxynitrite which in turn leads to 3-Nitrotyrosination with largely detrimental changes in protein function. Additionally, NO signalling alters protein function via S-nitrosylation. To date, little is known as to what extent these post-translational modifications contribute to or exacerbate neuronal dysfunction. We use glutamatergic synapses as a model system to identify novel nitrergic signalling pathways to correlate protein modifications with functional changes. The Drosophila neuromuscular junction was used to characterise NO effects employing electrophysiological methods. Two-electrode-voltage-clamp (TEVC) analyses were carried out in HL-3 solution using sharp electrodes (~30MΩ). Data denote mean±SEM (n-number) with *p<0.05 indicating statistical significance (t-test, ANOVA). TEVC data showed little NO (~0.1μM) effects on miniature excitatory junctional currents (mEJC) or decay kinetics but induced a reduction in mEJC frequencies (Ctrl: 1.6±0.1Hz (49) vs NO: 1.0±0.1Hz* (24)). Furthermore, evoked EJC amplitudes and quantal content were strongly reduced following NO exposure for >40min (Ctrl eEJC: 119±7nA (22) vs NO: 62±8nA* (14); Ctrl QC: 189±12 vs NO: 104±12*) suggestive for a reduction in presynaptic release. The above NO effects were detected following inhibition of the soluble guanylyl cyclase (sGC). Importantly, enhancing presynaptic S-Nitrosoglutathione reductase (GSNOR) enzyme activity, by overexpression (OE), prevents nitrergic effects. Cumulative postsynaptic current analysis (500ms 50Hz train) further showed a reduced number of release-ready vesicles following NO exposure (Ctrl: 276±21 (22) vs NO: 108±19* (14)) which was also confirmed by estimating the number of release sites using fluctuation analysis. Furthermore, pool sizes could be modulated in a positive or negative manner by enhancing or reducing GSNOR activity, respectively (Ctrl: OE: 212±25, RNAi: 139±23*; +NO: OE: 276±44, RNAi: 130±18*). Together, our data suggest that NO can modify synaptic signalling possibly via inducing post-translational protein modifications. This data interpretation is supported by the notion that sGC inhibition is ineffective but modulation of neuronal GSNOR activity impacts on synaptic physiology implying presynaptic actions of NO in a sGC-independent manner. The data extends our understanding of NO signalling, potentially leading to the identification of putative targets for therapeutic intervention(s) in disease.