Synaptic efficacy critically depends on the presynaptic intracellular calcium concentration ([Ca2+]i). We measured the calcium sensitivity of glutamate release in a rat auditory brainstem synapse by laser photolysis of caged calcium. A rise in [Ca2+]i to 1 micromolar readily evoked release. An increase to >30 micromolar depleted the releasable vesicle pool in <0.5 millisecond. A comparison with action potential-evoked release suggested that a brief increase of [Ca2+]i to approximately 10 micromolar would be sufficient to reproduce the physiological release pattern. Thus, the calcium sensitivity of release at this synapse is high, and the distinction between phasic and delayed release is less pronounced than previously thought.

Calcium entry through presynaptic calcium channels controls the release of neurotransmitter. It is not known whether the putative calcium sensor that triggers this rapid neurotransmitter release is close enough to be activated by the large increase in the Ca2+ concentration (calcium 'domain') reached within nanometres of a single calcium channel or whether many channels have to open. We tested this in a calyx-type synapse in the rat medial nucleus of the trapezoid body. We compared the quantal content of postsynaptic currents with the presynaptic calcium current that flows during an action potential, and the results suggest that more than 60 calcium channels open for each vesicle that is released. In addition, we dialysed terminals with the slow calcium buffer EGTA, which reduced phasic transmitter release at concentrations as low as 1 mM. These results indicate that the distance that calcium ions must diffuse to reach the calcium sensor is relatively long, and that therefore Ca2+ entry through multiple calcium channels is needed to release a vesicle.

1. Simultaneous whole-cell recordings in a rat brain slice preparation are described from presynaptic terminals (calyces of Held) and postsynaptic somata which form an axosomatic synapse in the medial nucleus of the trapezoid body (MNTB). 2. Presynaptic action potentials evoked suprathreshold excitatory postsynaptic potentials (EPSPs). The minimum synaptic delay was around 0 4 ms at 36°C and 0 9 ms at 23-24 0C. The amplitude of the L-ax-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptor-mediated component of the excitatory postsynaptic currents (EPSCs) was 2-13 nA (at -80 mV). 3. Current-voltage relations showed that presynaptic Ca2+ channels were of the high voltageactivated type. 4. A single action potential evoked a presynaptic fluorescence transient that decayed with a time constant of 0 3-0 7 s, depending on the concentration (60-200 #M) of the Ca2P indicator Calcium Green-5N (CG-5N). The peak amplitude of the [Ca2+]i transient was severalfold larger in the terminal than in the preterminal axon. 5. EPSC peak amplitudes were stable for more than 30 min after establishing the whole-cell configuration in the presynaptic terminal when the pipette contained 50/uM BAPTA. In contrast, with 1 mm BAPTA, peak amplitudes of EPSCs were reduced to one-third. 6. Trains of presynaptic action potentials evoked EPSCs with progressively smaller amplitudes. Little change was observed in the depression when the terminals were dialysed with 50/M BAPTA, whereas depression was reduced with 1 mm BAPTA. 7. In low (1 mM) [Ca2+]0, facilitation instead of depression of EPSCs was observed.8. The effects of presynaptic BAPTA suggest that the endogenous mobile Ca2P buffer capacity of giant presynaptic terminals in the MNTB is lower than in other terminals of fast transmitting synapses.Based on the homology of proteins involved in exocytosis in organisms as different as yeast and mice, release mechanisms are highly conserved during evolution. As specific models of how different presynaptic proteins interact to mediate neurotransmitter release are now emerging, it is desirable to test them under conditions that are as physiological as possible. The giant nerve ending of the squid stellate ganglion has been used successfully in the study of synaptic release mechanisms in invertebrates (reviewed in Katz, 1969). A mammalian preparation in which both the electrical properties of the presynaptic terminal and the composition of its cytoplasm can be manipulated, while at the same time having an estimate of its transmitter release, would make it possible to study release mechanisms in more detail than is currently possible in mammals. However, the small size of most presynaptic terminals in the mammalian central nervous system (CNS) Momiyama, 1993) and in the medial nucleus of the trapezoid body (MNTB; Forsythe, 1994). The MNTB participates in sound localization (Masterton, Jane & Diamond, 1967). Principal cells in the MNTB are innervated by large presynaptic terminals, the calyces of Held, that originate from cells in the contral...

The three-dimensional morphology of the axosomatic synaptic structures between a calyx of Held and a principal neuron in the medial nucleus of the trapezoid body (MNTB) in the brainstem of young postnatal day 9 rats was reconstructed from serial ultrathin sections. In the apposition zone between the calyx and the principal neuron two types of membrane specializations were identified: synaptic contacts (SCs) with active zones (AZs) and their associated postsynaptic densities (PSDs) constituted approximately 35% (n = 554) of the specializations; the remaining 65% (n = 1010) were puncta adherentia (PA). Synaptic contacts comprised approximately 5% of the apposition area of presynaptic and postsynaptic membranes. A SC had an average area of 0.100 microm(2), and the nearest neighbors were separated, on average, by 0.59 microm. Approximately one-half of the synaptic vesicles in the calyx were clustered within a distance of 200 nm of the AZ membrane area, a cluster consisting of approximately 60 synaptic vesicles (n = 52 SCs). Approximately two synaptic vesicles per SC were "anatomically docked." Comparing the geometry of the synaptic structure with its previously studied functional properties, we find that during a single presynaptic action potential (AP) (1) approximately 35% of the AZs release a transmitter quantum, (2) the number of SCs and anatomically docked vesicles is comparable with the low estimates of the readily releasable pool (RRP) of quanta, and (3) the broad distribution of PSD areas [coefficient of variation (CV) = 0.9] is likely to contribute to the large variability of miniature EPSC peaks. The geometry of the reconstructed synapse suggests that each of the hundreds of SCs is likely to contribute independently to the size and rising phase of the EPSC during a single AP.