Bursts of neuronal activity are transmitted more effectively as synapses mature. However, the mechanisms that control synaptic efficiency during development are poorly understood. Here, we study postnatal changes in synaptic ultrastructure and exocytosis in a calyx-type nerve terminal. Vesicle pool size, exocytotic efficiency (amount of exocytosis per Ca influx), Ca current facilitation, and the number of active zones (AZs) increased with age, whereas AZ area, number of docked vesicles per AZ, and release probability decreased with age. These changes led to AZs that are less prone to multivesicular release, resulting in reduced AMPA receptor saturation and desensitization. A greater multiplicity of small AZs with few docked vesicles, a larger pool of releasable vesicles, and a higher efficiency of release thus promote prolonged high-frequency firing in mature synapses.

Synaptogenesis during early development is thought to follow a canonical program whereby synapses increase rapidly in number and individual axons multiply-innervate nearby targets. Typically, a subset of inputs then out-competes all others through experience-driven processes to establish stable, long-lasting contacts. We investigated the formation of the calyx of Held, probably the largest nerve terminal in the mammalian CNS. Many basic functional and morphological features of calyx growth have not been studied previously, including whether mono-innervation, a hallmark of this system in adult animals, is established early in development. Evoked postsynaptic currents, recorded from neonatal mice between postnatal day 1 (P1) and P4, increased dramatically from Ϫ0.14 Ϯ 0.04 nA at P1 to Ϫ6.71 Ϯ 0.65 nA at P4 with sharp jumps between P2 and P4. These are the first functional assays of these nascent synapses for ages less than P3. AMPA and NMDA receptor-mediated currents were prominent across this age range. Electron microscopy (EM) revealed a concomitant increase, beginning at P2, in the prevalence of postsynaptic densities (16-fold) and adhering contacts (73-fold) by P4. Therefore, both functional and structural data showed that young calyces could form within 2 d, well before the onset of hearing around P8. Convergence of developing calyces onto postsynaptic targets, indicative of competitive processes that precede mono-innervation, was rare (4 of 29) at P4 as assessed using minimal stimulation electrophysiology protocols. Serial EM sectioning through 19 P4 cells further established the paucity (2 of 19) of convergence. These data indicate that calyces of Held follow a noncanonical program to establish targeted innervation that occurs over a rapid time course and precedes auditory experience.

The calyx of Held exhibits fast glutamatergic neurotransmission at high rates with low temporal jitter and has adapted specialized synaptic mechanisms to support its functional demands. We report the presence in calyces of an atypical arrangement of subcellular organelles, called the mitochondria-associated adherens complex (MAC). We demonstrate that MACs are located adjacent to synapses and contain membranous elements linked with coated and uncoated vesicles. Mitochondria that form MACs have more complex geometries than other mitochondria within the calyx and can extend between clusters of synapses. We estimate that the calyx contains 1600 MACs, 2400 synapses, and 6200 readily releasable vesicles. We also identify synaptic vesicle endocytotic regions close to MACs and synapses and hypothesize that calyces are composed of multiple activity modules, each containing machinery for vesicle release and recycling. The calyx of Held is one of the largest nerve terminals in the CNS (Held, 1893). Along with large and modified end-bulbs of Held delivered by auditory nerve fibers onto cochlear nucleus neurons (Cant and Morest, 1979;Tolbert and Morest, 1982;Fekete et al., 1984), these complex nerve terminals are key elements in brainstem circuitry that subserves sound localization (Morest, 1968). Spontaneous activity (generated in the absence of sound) can exceed 100 spikes/sec at the calyx terminal, and sound-driven activity at the most sensitive frequency of the calyceal neuron can approach 600 spikes/sec (Spirou et al., 1990). Temporal synchrony of calyceal neurons to a preferred phase of a low-frequency sound exceeds that found in the auditory nerve, and it can entrain (fire on every cycle of the stimulus sinusoid) at rates approaching 1 kHz (Joris et al., 1994a,b).High activity rates place demands on endocytotic mechanisms to maintain a pool of releasable vesicles. Some neurons, such as retinal bipolar cells, possess specialized structures called synaptic ribbons that are involved in vesicle trafficking, and therefore have unique mechanisms to solve their demands for synaptic activity (von Gersdorff and Matthews, 1999). It is plausible that the calyceal terminal, because of its extremely high spike rate and the temporal precision required to accomplish its task of sound localization, also uses unique structures and mechanisms.A noteworthy arrangement of organelles, consisting of a mitochondrion located near the presynaptic membrane and tethered via filaments to a punctum adherens, was first described in nerve terminals of the spinal cord (Gray, 1963). In the auditory system, this structure was noted in large and modified end-bulbs of auditory nerve fibers (Cant and Morest, 1979;Tolbert and Morest, 1982). More recently, we described this structure, which we named the mitochondria-associated adherens complex (MAC), in large collateral terminals of calyceal axons that are found in the superior olivary complex . Given the diversity of cellular elements that comprise the MAC, it could perform various functions in the nerve...

Hallmark features of neural circuit development include early exuberant innervation followed by competition and pruning to mature innervation topography. Several neural systems, including the neuromuscular junction and climbing fiber innervation of Purkinje cells, are models to study neural development in part because they establish a recognizable endpoint of monoinnervation of their targets and because the presynaptic terminals are large and easily monitored. We demonstrate here that calyx of Held (CH) innervation of its target, which forms a key element of auditory brainstem binaural circuitry, exhibits all of these characteristics. To investigate CH development, we made the first application of serial block-face scanning electron microscopy to neural development with fine temporal resolution and thereby accomplished the first time series for 3D ultrastructural analysis of neural circuit formation. This approach revealed a growth spurt of added apposed surface area (ASA) Ͼ200 m 2 /d centered on a single age at postnatal day 3 in mice and an initial rapid phase of growth and competition that resolved to monoinnervation in two-thirds of cells within 3 d. This rapid growth occurred in parallel with an increase in action potential threshold, which may mediate selection of the strongest input as the winning competitor. ASAs of competing inputs were segregated on the cell body surface. These data suggest mechanisms to select "winning" inputs by regional reinforcement of postsynaptic membrane to mediate size and strength of competing synaptic inputs.

Naϩ channel recovery from inactivation limits the maximal rate of neuronal firing. However, the properties of presynaptic Na ϩ channels are not well established because of the small size of most CNS boutons. Here we study the Na ϩ currents of the rat calyx of Held terminal and compare them with those of postsynaptic cells. We find that presynaptic Na ϩ currents recover from inactivation with a fast, single-exponential time constant (24°C, of 1.4 -1.8 ms; 35°C, 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 ϩ currents recover from inactivation with a doubleexponential time course ( fast of 1.2-1.6 ms; slow of 80 -125 ms; 24°C). Surprisingly, confocal immunofluorescence revealed that Na ϩ channels are mostly absent from the calyx terminal but are instead highly concentrated in an unusually long (Ϸ20 -40 m) unmyelinated axonal heminode. Outside-out patch recordings confirmed this segregation. Expression of Na v 1.6 ␣-subunit increased during development, whereas the Na v 1.2 ␣-subunit was not present. Serial EM reconstructions also revealed a long pre-calyx heminode, and biophysical modeling showed that exclusion of Na ϩ channels from the calyx terminal produces an action potential waveform with a shorter halfwidth. We propose that the high density and polarized locus of Na ϩ 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.