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, 23 (9), 3633-8

Developmental Increase in Vesicular Glutamate Content Does Not Cause Saturation of AMPA Receptors at the Calyx of Held Synapse

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Developmental Increase in Vesicular Glutamate Content Does Not Cause Saturation of AMPA Receptors at the Calyx of Held Synapse

Takayuki Yamashita et al. J Neurosci.

Abstract

Whether a quantal packet of transmitter saturates postsynaptic receptors is a fundamental question in central synaptic transmission. However, this question remains open with regard to saturation at mature synapses. The calyx of Held, a giant glutamatergic synapse in the auditory brainstem, becomes functionally mature during the fourth postnatal week in rats. During postnatal development, the mean amplitude of miniature (i.e., quantal) EPSCs (mEPSCs) becomes significantly larger. Experiments using the rapidly dissociating glutamate receptor antagonist kynurenate suggested that vesicular glutamate content increases with development. To test whether AMPA receptors are saturated by a packet of transmitter, we infused a high concentration of l-glutamate into mature calyceal terminals. This caused a marked increase in the mean amplitude of mEPSCs. We conclude that a single packet of transmitter glutamate does not saturate postsynaptic AMPA receptors even at the mature calyx of Held synapse with increased vesicular transmitter content.

Figures

Fig. 1.
Fig. 1.
Miniature EPSCs recorded from MNTB principal neurons in developing rats. Representative amplitude distributions of mEPSCs in P6, P14, P21, and P29 rats (number of events, 242, 370, 408, and 392) are shown with sample records (insets) of averaged mEPSCs (from all events of each distribution) (top inset) and superimposed records (4 traces) (bottom inset). Background noise distributions (filled bars) were obtained from the baselines of records with no clear events. Arrows indicate mean amplitude of mEPSCs. CV, Coefficient of variation (SD/mean) for the mEPSC amplitude.
Fig. 2.
Fig. 2.
Developmental changes in the amplitude, frequency, and kinetics of mEPSCs. Miniature EPSCs were recorded from the MNTB principal neurons in rats at P6–P7, P13–P14, P20–P21, and P28–P29 (n = 10 for each). A, The mean amplitude of mEPSCs increased from P13–P14 to P20–P21.B, The mean frequency of mEPSCs increased from P6–P7 to P20–P21. C, The 10–90% rise time of mEPSCs decreased from P6–P7 to P13–P14. Sample records (inset) are the rising phases of average mEPSCs normalized at the peak. D, The weighted mean of decay time constants decreased significantly between P6–P7 and P13–P14 and between P13–P14 and P20–P21. Sample records (inset) are the decay phases of average mEPSCs normalized at the peak.
Fig. 3.
Fig. 3.
Single-channel conductance of AMPA receptors underlying mEPSCs. Right column, Individual mEPSCs (top) (10 records superimposed after peak alignment) and averaged mEPSCs (bottom) at P6, P14, and P28. Left column, Peak-scaled variance-mean current plots obtained by the nonstationary fluctuation analysis for mEPSCs in P6, P14, and P28 rats. These ς2I plots were calculated from 162, 341, and 371 events, respectively. Mean single-channel current was estimated from the initial slope of the ς2I relationships. To estimate single-channel conductance (γ), reversal potentials of mEPSCs were measured in each recording.
Fig. 4.
Fig. 4.
Developmental increase in the quantal transmitter concentration in the synaptic cleft detected by the low-affinity AMPA receptor antagonist kynurenate (KYN) (50 μm).A, Sample records (averaged from 243 events for each) and amplitude histograms of mEPSCs recorded from an MNTB neuron, before (thin line) and after (thick line) application of kynurenate (superimposed), each at P14, P29, and P14 with 50 mml-glutamate (50Glu) loaded into a calyceal terminal. Filled bars are background noise histograms.B, C, Mean amplitude of mEPSCs (B) and their percentage inhibition by kynurenate (C) at P13–P14, P28–P29, and P13–P14 with 50 mml-glutamate loaded into calyceal terminals. Asterisks indicate a significant difference withp < 0.01 (Sheffe test).
Fig. 5.
Fig. 5.
Nonsaturation of postsynaptic AMPA receptors at mature synapses. A, Miniature EPSCs recorded from a P28 MNTB neuron before (i) and after (ii) loading 100 mml-glutamate into a calyceal terminal. Each data point represents the mean amplitude of mEPSCs sampled every 20 sec. Sample records of mEPSCs before (i) and after (ii) thel-glutamate loading at a slow sweep (top) and a fast sweep (bottom left, superimposed), and those normalized in the amplitude (bottom right, superimposed). B, Amplitude histograms of mEPSCs recorded from the same neuron before (i) and after (ii) thel-glutamate loading (number of events is 427 and 420, respectively). C, Summary data of mEPSCs from four synapses at P28–P29. The mean amplitude of mEPSCs before thel-glutamate loading was 54.3 ± 5.4 pA.D, Potentiation of evoked EPSCs by presynaptic loading of l-glutamate in a P29 rat. Sample records are averaged presynaptic action potentials (top, superimposed) and EPSCs before (i) and after (ii) the l-glutamate loading (bottom, superimposed). E, Summary data for evoked EPSCs (open circles) and spontaneous EPSCs (filled circles) from four synapses at P29. Before l-glutamate loading, the mean amplitude of evoked EPSCs was 4.37 ± 0.71 nA, and that of spontaneous EPSCs was 64.0 ± 10.6 pA. Glu, Glutamate. Dotted lines in Cand E denote mean values of data points beforel-glutamate loadings.

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