PROPERTIES OF TRANSMISSION AT A GIANT GLUTAMATERGIC SYNAPSE IN CEREBELLUM - THE MOSSY FIBER-UNIPOLAR BRUSH CELL SYNAPSE

1. The synaptic activation by messy fibers (MFs) of unipolar brush cells (UBCs) in the vestibular cerebellum (nodulus and uvula) was examined using patch-clamp recording methods in thin, rat cerebellar slices with Lucifer yellow-filled pipettes for subsequent fluorescence microscopic verification of the cell morphology. 2. UBCs were distinguished from adjacent granule cells in thin cerebellar slices in the uvula and nodulus regions by their larger soma diameters and short dendritic brush, greater whole-cell capacitance, and a prolonged, biphasic excitatory postsynaptic current (EPSC) to stimulation of MFs. 3. Thin-section transmission electron micrographs of the MF-UBC synapse displayed an unusually extensive area of synaptic apposition estimated to measure 12-40 mu m(2). The majority of UBCs was innervated by a single MF. At high magnification, individual clusters of presynaptic vesicles could be discerned, separated by regions of presynaptic membrane lacking vesicles, but apposed to continuous regions of postsynaptic density. Thus, after release, transmitter diffusion from the synaptic cleft must traverse considerable stretches of postsynaptic membrane before escape into extracellular space. In contrast, MF-granule cell synapses in these cerebellar regions resembled glutamate synapses in other brain regions in that the total synaptic area measured less than or equal to 4 mu m(2). These synaptic junctions were flanked by short stretches of unspecialized plasma membrane, providing a short (0.5 mu m) diffusional path from the site of neurotransmitter release to a branch point of the extracellular space. 4. The MF-evoked EPSC in UBCs was composed of a fast (10-90% rise time: 0.70 ms) and slow (10-90% rise time: 395 ms; 10-90% decay time: 3.1 s) component. The fast component was blocked by the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropopionic acid/kainate (AMPA/KA) antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (10 mu M) and displayed linear current-voltage (I-V) relations in the presence or absence of external magnesium. 5. The slow EPSC was also mediated by glutamate receptors, but in most neurons both AMPA/KA and N-methyl-D-aspartate (NMDA) receptors contributed to the slow EPSC, with the contribution of NMDA receptors predominating in the majority of cells. Consequently, although all cells displayed linear I-V relations in Mg2+ -free saline, cells in which the slow EPSC was predominently mediated by NMDA receptors exhibited voltage-dependent rectification in the presence of external Mg2+ (1 mM). 6. With increasing postnatal age (10-30 d), the contribution made to the slow EPSC by NMDA receptors declined, with a reciprocal increase in the contribution being made by AMPA/KA receptors. 7. The bath application of cyclothiazide (20-80 mu M), which blocks AMPA receptor desensitization, greatly enhanced the amplitude of the non-NMDA receptor-mediated component and smoothed the decay of the EPSC from its initial peak, indicating that desensitization sculpts both the time course and the absolute amplitude of the non-NMDA receptor-mediated component. 8. Both the fast and slow components of the MF-evoked EPSC were elicited in an all-or-none fashion at the same stimulus threshold, supporting anatomic data that UBCs receive innervation from single MFs. 9. In UBCs in which the axon was filled by Lucifer yellow, three-dimensional reconstructions of the cell using a laser-scanning confocal microscope revealed a branching axon of variable diameter that wandered within the granular layer without obvious orientation with respect to the plane of the folia and gave rise to two to four rosettelike terminals of large diameter (4-8 mu m). 10. Current-damp recordings of the excitatory postsynaptic potentials (EPSPs) evoked in UBCs by MF stimulation revealed an initial fast EPSP followed by a slow EPSP with a prolonged train of action potentials (0.5-2 s). With repetitive stimulation (1-10 Hz), the slow EPSPs summated to produce a depolarizing plateau for the duration of the stimulus train. 11. It is proposed that the prolonged time course of the slow EPSC results from an ultrastructural specialization at this giant glutamatergic synapse to entrap glutamate within the synaptic cleft after release. This results in a long-lasting synaptic potential that will produce a prolonged train of action potentials. Because UBCs are presumed to be glutamatergic, activation of the MFs innervating UBCs will result in a powerful feedforward excitation of target neurons within the granular layer. These neurons represent an important contribution to the basic cerebellar circuit in the vestibular regions of cerebellum where they are found in the highest density.