Prolonged Physiological Entrapment of Glutamate in the Synaptic Cleft of Cerebellar Unipolar Brush Cells

Abstract

The cellular mechanism underlying the genesis of the long-lasting alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-receptor-mediated excitatory postsynaptic currents (EPSCs) at the mossy fiber (MF)-unipolar brush cell (UBC) synapse in rat vestibular cerebellum was examined with the use of whole cell and excised patch-clamp recording methods in thin cerebellar slices. Activation of MFs evokes an all-or-none biphasic AMPA-receptor-mediated synaptic current with a late component that peaks at 100-800 ms, which has been proposed to originate from an entrapment of glutamate in the MF-UBC synaptic cleft and is generated by the steady-state activation of AMPA receptors. Bath application of cyclothiazide, which blocks desensitization of AMPA receptors, produced a dose-dependent enhancement of the amplitude of the synaptic current (median effective dose 30 microM) and slowing of the rise time of the fast EPSC. N-methyl-D-aspartate-receptor-mediated EPSCs in UBCs were not potentiated in amplitude or time course by cyclothiazide (100 microM). The dose-response relations for the steady-state current evoked by glutamate acting at AMPA receptors in excised outside-out patches from UBC and granule somatic membranes was biphasic, peaking at 50 microM and declining to 50-70% of this value at 1 mM glutamate. When glutamate was slowly washed from patches to simulate the gradual decline of glutamate in the synapse, a late hump in the transmembrane current was observed in patches from both cell types. The delivery of a second MF stimulus at the peak of the slow EPSC evoked a fast EPSC of reduced amplitude followed by an undershoot of the subsequent slow current, consistent with the hypothesis that the peak of the slow EPSC reflects the peak of the biphasic steady-state dose-response curve. Estimates of receptor occupancy and glutamate concentration derived from the ratio of fast EPSC amplitudes, and the amplitude and polarity of the initial steady-state current in paired-pulse experiments, predict a slow decline of glutamate with a time constant of 800 ms, declining to ineffective concentrations at 5.4 s. Manipulation of cleft glutamate concentration by lowered extracellular calcium or delivery of brief stimulus trains abolished the slow EPSC and restored the undershoot to paired stimuli, respectively, in a manner consistent with a prolonged lifetime of glutamate in the cleft. The slow component of the EPSC was prolonged in duration by the glutamate reuptake inhibitor L-trans-pyrrolidine-2,4-dicarboxylate, suggesting that glutamate transport contributes to the time course of the synaptic current in UBCs. The data support the notion that the MF-UBC synapse represents an ultrastructural specialization to effectively entrap glutamate for unusually prolonged periods of time following release from MF terminals. The properties of the postsynaptic receptors and constraints on diffusional escape of glutamate imposed by synaptic ultrastructure and glutamate transporters act in concert to sculpt the time course of the resulting ...

Variable timing of synaptic transmission in cerebellar unipolar brush cells

“…In rat UBCs, the initial fast EPSC is often followed by a transient increase of inward current, which rises to peak in hundreds of milliseconds (8)(9)(10). We reproduced this finding in mouse UBCs by applying a burst stimulation protocol, as shown in Fig.…”

“…Such slow resurgent EPSCs are mediated by AMPARs and are believed to arise from interplay between receptor desensitization and dose-response properties, due to long-lasting exposure to ambient glutamate (9). Earlier studies have generally considered the UBC EPSC as a stereotyped waveform (8)(9)(10)(11). It is the purpose of the current work to reexamine this assertion and characterize the temporal characteristics of slow UBC EPSCs.…”

“…At most central synapses, the lifetime of neurotransmitter in the cleft is short, limited by rapid diffusional escape to the surrounding medium (7). However, the glomerular structure and extensive synaptic apposition of UBC synapses have been hypothesized to promote prolonged entrapment of glutamate in the synaptic cleft (8,9).…”

“…In most UBCs, the biphasic appearance of EPSCs is particularly striking as the slow current first rises to peak in several hundreds of milliseconds, before decaying to baseline in seconds. Such slow resurgent EPSCs are mediated by AMPARs and are believed to arise from interplay between receptor desensitization and dose-response properties, due to long-lasting exposure to ambient glutamate (9). Earlier studies have generally considered the UBC EPSC as a stereotyped waveform (8)(9)(10)(11).…”

T-Type and L-Type Ca2+Conductances Define and Encode the Bimodal Firing Pattern of Vestibulocerebellar Unipolar Brush Cells

“…A key region for synaptic integration, the brush receives a single giant excitatory synapse, which is formed either by an extrinsic vestibular fiber or by the axon of another UBC (Jaarsma et al, 1995;Mugnaini et al, 1997;Nunzi et al, 2000Nunzi et al, , 2001. The presynaptic element of this synapse shows the typical ultrastructure of mossy fiber (mf) terminals but forms an exceptionally extended synaptic contact that can entrap glutamate for several hundred milliseconds, thus enabling unusually long excitatory responses (Rossi et al, 1995;Kinney et al, 1997). This unique input is tempered by the mixed GABAergic/glycinergic inhibition provided by Golgi cell terminals, impinging onto both the brush and the soma (Mugnaini et al, 1997;Dugué et al, 2005).…”