The density of AMPA receptors activated by a transmitter quantum at the climbing fibre-Purkinje cell synapse in immature rats.
We aimed to estimate the number of AMPA receptors (AMPARs) bound by the quantal transmitter packet, their single-channel conductance and their density in the postsynaptic membrane at cerebellar Purkinje cell synapses. The synaptic and extrasynaptic AMPARs were examined in Purkinje cells in 2- to 4-day-old rats, when they receive synaptic inputs solely from climbing fibres (CFs). Evoked CF EPSCs and whole-cell AMPA currents displayed roughly linear current-voltage relationships, consistent with the presence of GluR2 subunits in synaptic and extrasynaptic AMPARs. The mean quantal size, estimated from the miniature EPSCs (MEPSCs), was approximately 300 pS. Peak-scaled non-stationary fluctuation analysis of spontaneous EPSCs and MEPSCs gave a weighted-mean synaptic channel conductance of approximately 5 pS (approximately 7 pS when corrected for filtering). By applying non-stationary fluctuation analysis to extrasynaptic currents activated by brief glutamate pulses (5 mM), we also obtained a small single-channel conductance estimate for extrasynaptic AMPARs (approximately 11 pS). This approach allowed us to obtain a maximum open probability (Po,max) value for the extrasynaptic receptors (Po,max = 0.72). Directly resolved extrasynaptic channel openings in the continued presence of glutamate exhibited clear multiple-conductance levels. The mean area of the postsynaptic density (PSD) of these synapses was 0.074 microm2, measured by reconstructing electron-microscopic (EM) serial sections. Postembedding immunogold labelling by anti-GluR2/3 antibody revealed that AMPARs are localised in PSDs. From these data and by simulating error factors, we estimate that at least 66 AMPARs are bound by a quantal transmitter packet at CF-Purkinje cell synapses, and the receptors are packed at a minimum density of approximately 900 microm-2 in the postsynaptic membrane.
Momiyama
Akiko
A
Department of Pharmacology, University College London, UK. amomiyam@nips.ac.jp
Silver
R Angus
RA
Hausser
Michael
M
Notomi
Takuya
T
Wu
Yue
Y
Shigemoto
Ryuichi
R
Cull-Candy
Stuart G
SG
D000367:Age Factors; D000818:Animals; D002531:Cerebellum / Q000166:cytology / Q000254:growth & development / Q000502:physiology; D019706:Excitatory Postsynaptic Potentials / Q000502:physiology; D015640:Ion Channel Gating / Q000502:physiology; D016253:Microscopy, Immunoelectron; D009412:Nerve Fibers / Q000737:chemistry / Q000502:physiology / Q000648:ultrastructure; D009473:Neuronal Plasticity / Q000502:physiology; D009924:Organ Culture Techniques; D011689:Purkinje Cells / Q000737:chemistry / Q000166:cytology / Q000502:physiology*; D051381:Rats; D017207:Rats, Sprague-Dawley; D018091:Receptors, AMPA / Q000032:analysis / Q000502:physiology*; D013569:Synapses / Q000737:chemistry / Q000502:physiology* / Q000648:ultrastructure
D018091:Receptors, AMPA; C104723:glutamate receptor ionotropic, AMPA 3; C104722:glutamate receptor ionotropic, AMPA 2