Presynaptic Diversity Revealed by Ca²⁺-Permeable AMPA Receptors at the Calyx of Held Synapse

Abstract

GluA2-lacking Ca 2ϩ -permeable AMPARs (CP-AMPARs) play integral roles in synaptic plasticity and can mediate excitotoxic cellular signaling at glutamatergic synapses. However, the developmental profile of functional CP-AMPARs at the auditory brainstem remains poorly understood. Through a combination of electrophysiological and live-cell Ca 2ϩ imaging from mice of either sex, we show that the synaptic release of glutamate from the calyx of Held nerve terminal activates CP-AMPARs in the principal cells of the medial nucleus of the trapezoid body in the brainstem. This leads to significant Ca 2ϩ influx through these receptors before the onset of hearing at postnatal day 12 (P12). Using a selective open channel blocker of CP-AMPARs, IEM-1460, we estimate that ϳ80% of the AMPAR population are permeable to Ca 2ϩ at immature P4 -P5 synapses. However, after the onset of hearing, Ca 2ϩ influx through these receptors was greatly reduced. We estimate that CP-AMPARs comprise approximately 40% and 33% of the AMPAR population at P18 -P22 and P30 -P34, respectively. By quantifying the rate of EPSC block by IEM-1460, we found an increased heterogeneity in glutamate release probability for adult-like calyces (P30 -P34). Using tetraethylammonium (TEA), a presynaptic potassium channel blocker, we show that the apparent reduction of CP-AMPARs in more mature synapses is not a consequence of presynaptic action potential (AP) speeding. Finally, through postsynaptic AP recordings, we show that inhibition of CP-AMPARs reduces spike fidelity in juvenile synapses, but not in more mature synapses. We conclude that the expression of functional CP-AMPARs declines over early postnatal development in the calyx of Held synapse.The calyx of Held synapse is pivotal to the circuitry that computes sound localization. Postsynaptic Ca 2ϩ influx via AMPARs may be critical for signaling the maturation of this brainstem synapse. The GluA4 subunit may dominate the AMPAR complex at mature synapses because of its fast gating kinetics and large unitary conductance. The expectation is that AMPARs dominated by GluA4 subunits should be highly Ca 2ϩ permeable. However, we find that Ca 2ϩ -permeable AMPAR expression declines during postnatal development. Using the rate of EPSC block by IEM-1460, an open channel blocker of Ca 2ϩ -permeable AMPARs, we propose a novel method to determine glutamate release probability and uncover an increased heterogeneity in release probability for more mature calyces of Held nerve terminals.

Synaptic signals mediated by protons and acid‐sensing ion channels

“…This particular property of a giant synapse allows the recording of signals that may be present in other synapses but not resolved due to their small amplitude. We (Gonzalez-Inchauspe, Urbano, Guilmi, & Uchitel, 2017), have found that the presynaptic terminal of the brainstem calyx of Held synapse lacks ASIC channels while the MNTB neurons respond to acidification with a transient inward current sensitive to amiloride and to PcTx1(see also Lujan, Dagostin, & von Gersdorff, 2019).We have also shown the absence of these currents in ASIC1a −/− mice suggesting that homomeric ASIC-1a mediate these currents in MNTB neurons. Furthermore, we have detected ASIC1a-mediated currents during synaptic transmission after blocking AMPA and NMDA glutamate receptors, suggesting that an acidification of the synaptic cleft due to the co-release of neurotransmitter and H + from synaptic vesicles is capable of activating ASIC postsynaptic channels.…”

“…Currently, the time course of pH changes at fast synapses has not been measured in detail, but from indirect estimations at ribbon‐type and hippocampal synapses (Du et al, ; Palmer et al, ), it is possible to hypothesize that the synaptic cleft pH waveform is complex, including an acid and alkaline shift with a time course that depends on the buffer capacity of the cleft. The resulting change in extracellular pH is, therefore, likely to be much smaller than at ribbon synapses but sufficient to inhibit AMPA receptors by enhancing desensitization (Lei, Orser, Thatcher, Reynolds, & MacDonald, ; Traynelis & Cull‐Candy, ); and to activate ASICs and generate a postsynaptic current as shown by Du et al () at the amygdala; Kreple et al () at the nucleus accumbens and by our group at the mouse calyx of Held (Gonzalez‐Inchauspe, Urbano, Di Guilmi, & Uchitel, ), confirmed recently by Lujan, Dagostin, and von Gersdorff (), (Figure [left]). Furthermore, cleft pH can also remain acidic during prolonged stimulation or during a variety of pathological conditions such as during epileptic activity.…”

“…This particular property of a giant synapse allows the recording of signals that may be present in other synapses but not resolved due to their small amplitude. We (Gonzalez‐Inchauspe, Urbano, Guilmi, & Uchitel, ), have found that the presynaptic terminal of the brainstem calyx of Held synapse lacks ASIC channels while the MNTB neurons respond to acidification with a transient inward current sensitive to amiloride and to PcTx1 (see also Lujan, Dagostin, & von Gersdorff, ). We have also shown the absence of these currents in ASIC1a −/− mice suggesting that homomeric ASIC‐1a mediate these currents in MNTB neurons.…”

“…The small ASIC synaptic currents recorded after full blockade are shown as raw data and as an average of 20 samples. ASIC synaptic currents recorded from another MNTB neuron were potentiated by histamine and suppressed by increasing the buffer capacity of the media with Hepes 10 mM (lower part of the panel) (Gonzalez‐Inchauspe et al, , in press; Lujan et al, ). Right side: Immunohistochemistry performed on a slice containing the MNTB nucleus from a mouse expressing S100 GFP to label astrocytes.…”

“…It is now well established that in some synapses, protons activate an inward current at postsynaptic cells. However, the amplitude of the proton‐activated EPSCs is small, 1%–5%, compared to that generated by glutamate gating AMPA receptors (Du et al, ; Gonzalez‐Inchauspe et al, ; Highstein et al, ; Kreple et al, ; Lujan et al, ). Despite the small amplitude and the inhibitory effect of protons on NMDA receptors, ASIC activation allows Na + influx, postsynaptic depolarization and the relief of blockade of NMDA receptors by Mg 2+ , thus, facilitating Ca 2+ influx through them.…”