Motor axons and their terminals undergo Wallerian degeneration (WD) following nerve injury. Here we asked whether neuromuscular activity increases vulnerability of motor axons or provides protection against WD. We used Wlds mice in which WD is about ten times slower than normal (Lunn et al. 1989, Gillingwater et al., 2002). Glass capillaries containing 15mM TTX were implanted under general anaesthesia (isoflurane and medical oxygen, 0.4 litres min-1; Martinov and Nja 2005). A plastic cuff secured the tip adjacent to the sciatic nerve. The efficacy of nerve block was checked daily for the following 7 days by monitoring toe-pinch reflex and grip strength. Nerve stimulation in anaesthetised mice confirmed the patency of the nerve block. One week post-implant the animals were re-anaesthetised and the tibial nerve was cut. Five days post-axotomy, intracellular recordings of spontaneous MEPPs and evoked EPPs were made in isolated flexor digitorum brevis (FDB) muscles in which action potentials were abolished with µ-conotoxin (2 µM). Preconditioning with TTX-block resulted in a decrease in the level of WldS-induced synaptic protection compared with axotomised saline-treated or unoperated controls (8.57±2.51% responsive fibres, n=7, vs. 55.56±5.88% n=3, vs. 43.34±10.11% n=6 respectively, p<0.05 one way ANOVA). TTX treatment or microcapsule implantation alone did not cause any detectable damage: electrophysiological analysis showed almost all fibres responding with EPPs (87.78±7.78% n=3 TTX-blocked; 85.00±2.15% n=4 saline-treated; 91.88±2.83% n=6 unoperated controls, p>0.05 one way ANOVA). Repeated visualisation of axons in vivo in thy1.2:YFP16-Wlds mice by confocal microendoscopy (Wong et al., 2009) showed no discernible effect of TTX block on the protection of axons by WldS. Preconditioning block in C57Bl6 mice also showed a reduced number of fibres responding to stimulation 12h post-axotomy compared to axotomised controls (6.25±3.75% n=4, vs. 61.67±17.56% n=4 respectively, p<0.01 unpaired t-test). To test for a direct influence of activity on synaptic degeneration, isolated FDB nerve-muscle preparations were maintained in oxygenated mammalian saline at 32 °C for 12-48 hours. Electrophysiological analysis proved that this method adequately differentiates the rates of synaptic degeneration in wild-type and Wlds preparations. After 5-15h ex vivo, fewer fibres were responsive to stimulation in WT muscles than Wlds (36.39±16.02% n=6, vs. 89.61±2.93% n=11, p<0.05 unpaired t-test). Intermittent high frequency (100Hz, for 1 second every 100 seconds) enhanced synaptic degeneration, in comparison to constant low frequency (1Hz) stimulation (78.75±11.21% responsive fibres, n=4, vs. 32.67±6.86% n=4, p<0.05 unpaired t-test). The data provide evidence that vulnerability of neuromuscular synapses to stimuli that trigger degeneration is selectively enhanced by axonal disuse.