Group III metabotropic glutamate receptors (mGluRs) are expressed at the calyx of Held, a large glutamatergic presynaptic terminal in the auditory brainstem. Pharmacological activation of these receptors results in reduction of the presynaptic calcium current and inhibition of neurotransmitter release (Takahashi et al. 1996, Science 274:594). Despite this, endogenous glutamate release appears to have little effect on neurotransmission, questioning the role of these mGluRs as autoreceptors (von Gersdorff et al. 1997, J Neurosci 17:8137). However, closer examination of the EPSCs during and after high frequency trains reveals autoreceptor mediated depression of release probability. Brainstem slices (200μm thick) were prepared from Lister Hooded rats (P10-14), humanely killed by decapitation. Postsynaptic medical nucleus of the trapezoid body neurons were whole-cell voltage-clamped and EPSCs were elicited by electrical stimulation of the presynaptic axon. EPSCs were recorded at 37⁰C, in the presence of 2mM kynurenate to reduce receptor saturation and desensitization. Data are mean±SEM of 5 cells. Application of 50μM L-(+)-2-amino-4-phosphonobutyric acid (L-AP4), a specific group III mGluR agonist, reduced EPSC amplitude by 79±3%. This inhibition was reversed by the competitive mGluR antagonist (R,S)-cyclopropyl-4-phosphonophenylglycine (CPPG, 300μM) indicating that this concentration of CPPG would be sufficient to antagonise the action of endogenously released glutamate. To mimic physiological levels of communication at this synapse, 200Hz stimulation trains were applied for 1s. The EPSC amplitude rapidly depressed to a steady state, which was unaffected by CPPG, suggesting no apparent mGluR activation by released glutamate. However, the paired-pulse ratio of two EPSCs evoked 1s after the train changed from 1.18±0.06 (facilitating) to 0.97±0.03 (depressing; P=0.01, paired t-test) in CPPG. This result shows that activation of presynaptic mGluRs by endogenously released glutamate lowers release probability. A simple model of vesicle exocytosis reveals that changes in release probability during a synaptic train are passively compensated by changes in the size of the readily releasable pool such that exocytosis remains constant. This means that the level of EPSC depression is not a good indicator of mGluR autoreceptor activation since reduced release probability will conserve readily releasable vesicles. These results reveal that presynaptic mGluR receptors are activated during physiological trains of activity thereby influencing short-term plasticity. Supported by The Wellcome Trust and The Royal Society.