Train stimulation of parallel fibre to Purkinje cell inputs reveals two populations of synaptic responses with different receptor signatures

Abstract

Key pointsr Purkinje cells of the cerebellum receive ß180,000 parallel fibre synapses, which have often been viewed as a homogeneous synaptic population and studied using single action potentials.r Many parallel fibre synapses might be silent, however, and granule cells in vivo fire in bursts.Here, we used trains of stimuli to study parallel fibre inputs to Purkinje cells in rat cerebellar slices.r Analysis of train EPSCs revealed two synaptic components, phase 1 and 2. Phase 1 is initially large and saturates rapidly, whereas phase 2 is initially small and facilitates throughout the train. The two components have a heterogeneous distribution at dendritic sites and different pharmacological profiles.r The differential sensitivity of phase 1 and phase 2 to inhibition by pentobarbital and NBQX mirrors the differential sensitivity of AMPA receptors associated with the transmembrane AMPA receptor regulatory protein, γ-2, gating in the low-and high-open probability modes, respectively.Abstract Cerebellar granule cells fire in bursts, and their parallel fibre axons (PFs) form ß180,000 excitatory synapses onto the dendritic tree of a Purkinje cell. As many as 85% of these synapses have been proposed to be silent, but most are labelled for AMPA receptors. Here, we studied PF to Purkinje cell synapses using trains of 100 Hz stimulation in rat cerebellar slices. The PF train EPSC consisted of two components that were present in variable proportions at different dendritic sites: one, with large initial EPSC amplitude, saturated after three stimuli and dominated the early phase of the train EPSC; and the other, with small initial amplitude, increased steadily throughout the train of 10 stimuli and dominated the late phase of the train EPSC. The two phases also displayed different pharmacological profiles. Phase 2 was less sensitive to inhibition by NBQX but more sensitive to block by pentobarbital than phase 1. Comparison of synaptic results with fast glutamate applications to recombinant receptors suggests that the high-open-probability gating mode of AMPA receptors containing the auxiliary subunit transmembrane AMPA receptor regulatory protein γ-2 makes a substantial contribution to phase 2. We argue that the two synaptic components arise from AMPA receptors with different functional signatures and synaptic distributions. Comparisons of voltage-and current-clamp responses obtained from the same Purkinje cells indicate that phase 1 of the EPSC arises from synapses ideally suited to transmit short bursts of action potentials, whereas phase 2 is likely to arise from low-release-probability or 'silent' synapses that are recruited during longer bursts. Abbreviations AMPAR, AMPA receptor; D-AP5, D-(−)-2-amino-5-phosphonopentanoic acid; CNQX, 6-cyano-7-nitroquinoxaline-2,3-dione; CPCCOEt, 7-(hydroxyimino)cyclopropa [b]chromen-1a-carboxylate ethyl ester; GYKI 53655, 1-(4-aminophenyl)-3-methylcarbamyl-4-methyl-7,8-methylenedioxy-3,4-dihydro-5H-2,3-benzodiazepine; I ss , steady-state current; mGluR1, metabotropic glutamate recepto...

Mechanisms of postsynaptic localization of AMPA-type glutamate receptors and their regulation during long-term potentiation

“…These studies raise interesting questions about the essential requirement for TARPs in AMPAR forward trafficking and the percentage of receptors that are TARP associated throughout their life cycle. Different populations of synapses, suggested to contain different levels of TARP association, have been recorded in Purkinje neurons of the cerebellum (35); however, PDZ interactions of TARPs appear to be essential for all AMPAR postsynaptic anchoring in hippocampal CA1 cells (36).…”

Control of AMPA Receptor Activity by the Extracellular Loops of Auxiliary Proteins

“…Both these signatures of activity should be useful tools for investigating TARP action in synapses, including understanding the relative importance of assembly into complexes for anchoring (Opazo et al, 2010) as opposed to kinetic modulation, for clarifying the consequences of TARP modulation for short term plasticity (Devi et al, 2016), and for better identifying TARPs in ternary complexes with other auxiliary subunits (Khodosevich et al, 2014;Herring et al, 2013).…”

“…The physiological importance of modulation is likely to be the specialization of particular codes of short-term plasticity, in the hippocampus and cerebellum at least (von Engelhardt et al, 2010;Klaassen et al, 2016;Khodosevich et al, 2014;Devi et al, 2016). Recently, antagonists of AMPA receptors that target GluA2-g8 complexes were described (Maher et al, 2016;Kato et al, 2016), further enhancing interest in the molecular basis of complexes of GluA subunits and their auxiliary proteins as potential drug targets.…”

Control of AMPA receptor activity by the extracellular loops of auxiliary proteins

“…This work has produced mutant TARPs and AMPA receptors that both lack modulatory properties, and also those that have greatly enhanced modulation. Both these signatures of activity should be useful tools for investigating TARP action in synapses, including understanding the relative importance of assembly into complexes for anchoring (39) as opposed to kinetic modulation, for clarifying the consequences of TARP modulation for short term plasticity (18), and for better identifying TARPs in ternary complexes with other auxiliary subunits (17, 40).…”

“…TARPs and other auxiliary proteins modify the gating and pharmacology of synaptic AMPA receptors (14, 15). The physiological importance of modulation is likely to be the specialization of particular codes of short-term plasticity, in the hippocampus and cerebellum at least (7, 16–18). Recently, antagonists of AMPA receptors that target GluA2–γ8 complexes were described (19, 20), further enhancing interest in the molecular basis of complexes of GluA subunits and their auxiliary proteins as potential drug targets.…”