br Introduction AMPA receptors AMPARs mediate the majority o

Introduction AMPA receptors (AMPARs) mediate the majority of fast excitatory postsynaptic currents (EPSCs) in the perifosine mg (Jonas, 2000). The brevity of EPSCs and rapid deactivation of AMPARs depend upon a short lifetime of synaptically released glutamate, estimated to be about 1 ms (Clements et al., 1992). Another key factor contributing to fast AMPAR-mediated EPSCs is rapid desensitization, which decreases response amplitudes by >90% within ∼10 ms upon prolonged exposure to glutamate (Colquhoun et al., 1992, Raman and Trussell, 1992, Silver et al., 1996, Trussell and Fischbach, 1989). Thus, even when cleft glutamate clearance is slow, desensitization still forces AMPAR-mediated EPSCs to decay quickly (Trussell et al., 1993). In stark contrast to this picture of typical AMPAR synapses is the large mossy fiber-unipolar brush cell (UBC) synapse in the granular layer of cerebellar cortex and cochlear nucleus (Floris et al., 1994, Rossi et al., 1995). Stimulation of this synapse evokes typical fast EPSCs, but these are followed by a slow, AMPAR-mediated EPSC lasting hundreds of milliseconds (Borges-Merjane and Trussell, 2015, Kinney et al., 1997, Rossi et al., 1995). The mossy fiber-UBC synapse features an extensive, convoluted synaptic cleft between the presynaptic terminal and postsynaptic brush-like dendrite (Rossi et al., 1995). Kinney et al. (1997) proposed that the slow current is the combined result of delayed clearance from this large synaptic cleft and the biophysical properties of AMPARs. Upon such prolonged glutamate exposure, synaptic AMPARs would enter steady-state desensitization and occasionally reopen, generating the slow EPSC. However, direct evidence for “glutamate entrapment” (Rossi et al., 1995) requires information about the kinetic state of receptors during synaptic transmission, their molecular properties, and the forces that determine the glutamate lifetime in the cleft. We tested this hypothesis in UBCs of the vestibular cerebellum. Fast UV uncaging of glutamate after synaptic stimuli revealed that after exocytosis of glutamate, >90% of AMPARs become desensitized. Thereafter, receptors slowly recover from desensitization concurrent with an increase in the EPSC amplitude. Dose-response relations show that AMPARs produce smaller equilibrium responses to millimolar levels of glutamate than to micromolar levels, suggesting that the slow EPSC tracks recovery from desensitization as glutamate is removed. This decrease in response to high glutamate levels was absent in UBCs from γ2 transmembrane AMPAR regulatory protein (TARP) mutant stargazer (stg) mice, and slow EPSCs in these mice were reduced in amplitude. Finally, the slow time course of glutamate was dictated by glutamate transporters, as block of transport profoundly distorted synaptic responses. Thus, at the mossy-fiber-UBC synapse, exocytosis initiates a process of transmission controlled by a balance between glutamate uptake and the heightened sensitivity to transmitter bestowed by TARPs.
Results
Discussion This study showed that the slow EPSC of UBCs is determined by two factors. Continual transporter activity controls the time course of the EPSC, the depth of desensitization, and the baseline level of glutamate in the synaptic cleft. Moreover, a TARP-containing AMPAR renders the cell sensitive to the slowly changing levels of glutamate established by the transporters. In the absence of transport, glutamate levels fall extremely slowly and never reach a level below the sensitivity of the AMPAR, highlighting the “entrapment” hypothesis originally proposed by Rossi et al. (1995). This situation is dramatically different from that of mossy fiber synapses made onto granule cells or of fenestrated calyceal synapses, both of which have exclusively fast phasic AMPAR-mediated transmission even after transporter blockade (Renden et al., 2005, Sylantyev et al., 2013); thus, entrapment, transporter action, and the AMPAR-TARP complex allow the UBC to transmit in a nearly tonic mode despite sensing phasic exocytosis.