We are examining the role of Transforming Growth Factor-β2 (TGF-β2) at mammalian synapses. TGF-β2 is the most highly expressed cytokine in the healthy adult mammalian nervous system. It is particularly expressed in skeletal muscle fibres at neuromuscular junctions (NMJs), while motor terminals express the high affinity receptor for TGF-β2 binding (McLennan & Koishi, 2002). We recently showed TGF-β2 enhances neuromuscular transmission by increasing loading of neurotransmitter into synaptic vesicles (increasing quantal size; Fong et al, 2010). It also increases evoked postsynaptic potentials, but reduces the number of vesicles released per action potential (lower quantum content). These effects occur with 60 min pre-incubation at 1ng/ml. Thus, TGF-β2 acutely increases both efficacy (bigger potentials) and efficiency (fewer vesicles required). However, it is still unclear if endogenous TGF-β2 can be released as a retrograde modulator of neuromuscular transmission. Adult Sprague-Dawley rats were humanely killed by cervical dislocation (Schedule 1, ASPA 1986) and the amplitudes of spontaneous (miniature end-plate potentials, mEPPs) and nerve-stimulation evoked ACh release (EPPs) recorded from NMJs in excised hemidiaphragm/phrenic nerve preparations with sharp intracellular microelectrodes. µ-conotoxin GIIIB blocked muscle contraction. EPP amplitude rundown during 20 Hz, 2 min trains was also assessed, comparing first and subsequent trains applied one hour later. Data are expressed as mean ± SE, n (NMJs) from 6-15 preparations and compared by Student’s t-test (means) or 2-way ANOVA (trains). As previously (Fong et al, 2010), 1ng/ml TGF-β2 significantly increased amplitudes of mEPPs (0.50 ± 0.06 mV, 14 to 0.70 ± 0.03 mV, 22; p<0.01) and EPPs (24.3 ± 1.9 mV, 14 to 28.5 ± 1.1 mV, 22; p<0.05). Conversely, TGF-β2 neutralising antibody reduced the size of mEPPs (0.32 ± 0.04 mV, 15; p<0.02) and EPPs (18.2 ± 1.5 mV, 15; p<0.02). TGF-β2 significantly increased EPP amplitudes throughout a train (n=15 & 6; p<0.0001). Strikingly, in control muscles given a second train 1 hr later EPPs were also larger (n=8, p<0.0001), and not significantly different from a first EPP train in TGF-β2 (p=0.3). The effect of repeated trains was abolished by neutralising antibodies to TGF-β2 (n=5 & 8; p=0.8), while repeated trains in TGF-β2 produced only a small further increase (n=15, p=0.02). These data indicate nerve activity trains release endogenous TGF-β2 from postsynaptic muscle fibres that enhances subsequent neurotransmission. Thus, the data indicate TGF-β2 is an activity-dependent retrograde modulator of synaptic transmission.