The timing and strength of synaptic transmission is profoundly dependent on temperature. However, the temperature dependence of the multiple mechanisms that contribute to short-term synaptic plasticity is poorly understood. Here, we use voltage-clamp recordings to quantify the temperature dependence of exocytosis at the calyx of Held synapse. EPSC and miniature EPSC amplitudes were larger at physiological temperature, but quantal content during low-frequency (0.05 Hz) stimulation was constant after temperature jumps from 22–24°C to 35–37°C. The initial degree of EPSC depression during 100 Hz stimuli trains was unchanged with temperature, as were estimates of release probability and vesicle pool size. In contrast, physiological temperatures dramatically relieved depression measured after 40 stimuli at 100 Hz by increasing twofold the rate of recovery from depression. Presynaptic calyx recordings revealed that physiological temperature increased capacitance jumps resulting from 0.5 and 1 ms depolarizations by increasing Ca²⁺influx. When Ca²⁺entry was equalized at the two temperatures, exocytosis exhibited little temperature dependence for brief depolarizations. However, in response to longer depolarizations, raising temperature increased a slow phase of exocytosis, without affecting overall Ca²⁺entry or the size of the readily releasable pool of vesicles. Higher temperatures also increased the rate of presynaptic Ca²⁺current inactivation; nevertheless, the degree of steady-state EPSC depression was greatly reduced. Our results thus suggest that changes in steady-state EPSCs during stimulus trains at physiological temperature reflect larger quantal amplitudes and faster refilling of synaptic vesicle pools, leading to reduced short-term depression during prolonged high-frequency firing.

Synaptic vesicle membrane must be quickly retrieved and recycled after copious exocytosis to limit the depletion of vesicle pools. The rate of endocytosis at the calyx of Held nerve terminal has been measured directly using membrane capacitance measurements from immature postnatal day P7–P10 rat pups at room temperature (RT: 23–24°C). This rate has an average time constant of tens of seconds and becomes slower when the amount of exocytosis (measured as capacitance jump) increases. Such slow rates seem paradoxical for a synapse that can operate continuously at high-input frequencies. Here we perform time-resolved membrane capacitance measurements from the mouse calyx of Held in brain stem slices at physiological temperature (PT: 35–37°C), and also from more mature calyces after the onset of hearing (P14–P18). Our results show that the rate of endocytosis is strongly temperature dependent, whereas the endocytotic capacity of a nerve terminal is dependent on developmental stage. At PT we find that endocytosis accelerates due to the addition of a kinetically fast component (time constant: τ = 1–2 s) immediately after exocytosis. Surprisingly, we find that at RT the rate of endocytosis triggered by short (1- to 5-ms) or long (≥10-ms) depolarizing pulses in P14–P18 mice are similar (τ ≈ 15 s). Furthermore, this rate is greatly accelerated at PT (τ ≈ 2 s). Thus endocytosis becomes faster and less saturable during synaptic maturation, making the calyceal terminal more capable of sustaining prolonged high-frequency transmitter release.

Na⁺channel recovery from inactivation limits the maximal rate of neuronal firing. However, the properties of presynaptic Na⁺channels are not well established because of the small size of most CNS boutons. Here we study the Na⁺currents of the rat calyx of Held terminal and compare them with those of postsynaptic cells. We find that presynaptic Na⁺currents recover from inactivation with a fast, single-exponential time constant (24°C, τ of 1.4-1.8 ms; 35°C, τ of 0.5 ms), and their inactivation rate accelerates twofold during development, which may contribute to the shortening of the action potential as the terminal matures. In contrast, recordings from postsynaptic cells in brainstem slices, and acutely dissociated, reveal that their Na⁺currents recover from inactivation with a double-exponential time course (τ_fastof 1.2-1.6 ms; τ_slowof 80-125 ms; 24°C). Surprisingly, confocal immunofluorescence revealed that Na⁺channels are mostly absent from the calyx terminal but are instead highly concentrated in an unusually long (≈20-40 μm) unmyelinated axonal heminode. Outside-out patch recordings confirmed this segregation. Expression of Na_v1.6 α-subunit increased during development, whereas the Na_v1.2α-subunit was not present. Serial EM reconstructions also revealed a long pre-calyx heminode, and biophysical modeling showed that exclusion of Na⁺channels from the calyx terminal produces an action potential waveform with a shorter half-width. We propose that the high density and polarized locus of Na⁺channels on a long heminode are critical design features that allow the mature calyx of Held terminal to fire reliably at frequencies near 1 kHz.