Silent Synapses in Developing Cerebellar Granule Neurons

Abstract

Silent synapses are excitatory synapses endowed exclusively with N-methyl-d-aspartate (NMDA) responses that have been proposed to acquire α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) responses during development and after long-term potentiation (LTP). These synapses are functionally silent because of the Mg2+ block of NMDA receptors at resting potentials. Here we provide evidence for the presence of silent synapses in developing cerebellar granule cells. Using the patch-clamp technique in the whole-cell configuration, we recorded the spontaneous excitatory postsynaptic currents (sEPSCs) from rat cerebellar granule cells in culture and in slices at physiological concentration of Mg2+ (1 mM). A holding potential of +60 mV removes Mg2+ block of NMDA channels, allowing us to record NMDA-sEPSCs. We thus compared the frequency of AMPA-sEPSCs, recorded at −60 mV, with that of NMDA-sEPSCs, recorded at +60 mV. NMDA-sEPSCs occurred at higher frequency than the AMPA-sEPSCs in most cells recorded in slices from rats at postnatal day (P) <13 and in culture at 6–8 days after plating (DIV6–8). In a few cells from young rats (P6–9) and in most neurons in culture at DIV6 we recorded exclusively NMDA-sEPSCs, supporting the hypothesis of existence of functional synapses with NMDA and without AMPA receptors. Increasing glutamate release in the slice with cyclothiazide and temperature increased AMPA and NMDA-sEPSCs frequencies but failed to alter the relative ratio of frequency of occurrence. Frequency ratio of NMDA versus AMPA-sEPSCs in slices was correlated with the weighted time constant of decay (τ w ) of NMDA-sEPSCs and decreased with development along the reported decrease of τ w . We suggest that the prevalence of synaptic NR2A subunits that confer faster kinetics is paralleled by the disappearance of silent synapses early in cerebellar development.