PROLONGED PHYSIOLOGICAL ENTRAPMENT OF GLUTAMATE IN THE SYNAPTIC CLEFTOF CEREBELLAR UNIPOLAR BRUSH CELLS

The cellular mechanism underlying the genesis of the long-lasting lpha -amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-receptor-me diated excitatory postsynaptic currents (EPSCs) al the messy fiber(MF) -unipolar brush cell (UBC) synapse in rat vestibular cerebellum was ex amined with the use of whole cell and excised patch-clamp recording me thods in thin cerebellar slices. Activation of MFs evokes an all-or-no ne biphasic AMPA-receptor-mediated synaptic current with a late compon ent that peaks at 100-800 ms, which has been proposed to originate fro m an entrapment of glutamate in the MF-UBC synaptic cleft and is gener ated by the steady-state activation of AMPA receptors. Bath applicatio n of cyclothiazide, which blocks desensitization of AMPA receptors, pr oduced a dose-dependent enhancement of the amplitude of the synaptic c urrent (median effective dose 30 mu M) and slowing of the rise time of the fast EPSC. N-methyl-D-aspartate-receptor mediated EPSCs in UBCs w ere not potentiated in amplitude or time course by cyclothiazide (100 mu M). 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 mu M and declining to 50-70% of this value at 1 mM glutamate. When glutam ate 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 a mplitude followed by an undershoot of the subsequent slow current, con sistent with the hypothesis that the peak of the slow EPSC reflects th e 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 decl ine of glutamate with a time constant of 800 ms, declining to ineffect ive concentrations al 5.4 s. Manipulation of cleft glutamate concentra tion by lowered extracellular calcium or delivery of brief stimulus tr ains abolished the slow EPSC and restored the undershoot to paired sti muli, respectively, in a manner consistent with a prolonged lifetime-o f 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 t he time course of the synaptic current in UBCs. The data support the n otion that the MF-UBC synapse represents an ultrastructural specializa tion to effectively entrap glutamate for unusually prolonged periods o f time following release from MF terminals. The properties of the post synaptic receptors and constraints on diffusional escape of glutamate imposed by synaptic ultrastructure and glutamate transporters act in c oncert to sculpt the time course of the resulting slow EPSC. This in t urn drives a long-lasting train of action potentials in response to si ngle presynaptic stimuli.