George A. Spirou

Optimizing Synaptic Architecture and Efficiency for High-Frequency Transmission

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.

Presynaptic Na+ Channels: Locus, Development, and Recovery from Inactivation at a High-Fidelity Synapse

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 (τfast of 1.2-1.6 ms; τslow of 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 Nav1.6 α-subunit increased during development, whereas the Nav1.2α-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.

Synaptogenesis of the Calyx of Held: Rapid Onset of Function and One-to-One Morphological Innervation

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 −0.14 ± 0.04 nA at P1 to −6.71 ± 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.

Human Cortical Organization for Processing Vocalizations Indicates Representation of Harmonic Structure as a Signal Attribute

The ability to detect and rapidly process harmonic sounds, which in nature are typical of animal vocalizations and speech, can be critical for communication among conspecifics and for survival. Single-unit studies have reported neurons in auditory cortex sensitive to specific combinations of frequencies (e.g., harmonics), theorized to rapidly abstract or filter for specific structures of incoming sounds, where large ensembles of such neurons may constitute spectral templates. We studied the contribution of harmonic structure to activation of putative spectral templates in human auditory cortex by using a wide variety of animal vocalizations, as well as artificially constructed iterated rippled noises (IRNs). Both the IRNs and vocalization sounds were quantitatively characterized by calculating a global harmonics-to-noise ratio (HNR). Using functional MRI, we identified HNR-sensitive regions when presenting either artificial IRNs and/or recordings of natural animal vocalizations. This activation included regions situated between functionally defined primary auditory cortices and regions preferential for processing human nonverbal vocalizations or speech sounds. These results demonstrate that the HNR of sound reflects an important second-order acoustic signal attribute that parametrically activates distinct pathways of human auditory cortex. Thus, these results provide novel support for the presence of spectral templates, which may subserve a major role in the hierarchical processing of vocalizations as a distinct category of behaviorally relevant sound.

The Micro-Architecture of Mitochondria at Active Zones: Electron Tomography Reveals Novel Anchoring Scaffolds and Cristae Structured for High-Rate Metabolism

Mitochondria are integral elements of many nerve terminals. They must be appropriately positioned to regulate microdomains of Ca2+ concentration and metabolic demand, but structures that anchor them in place have not been described. By applying the high resolution of electron tomography (ET) to the study of a central terminal, the calyx of Held, we revealed an elaborate cytoskeletal superstructure that connected a subset of mitochondria to the presynaptic membrane near active zones. This cytoskeletal network extended laterally and was well integrated into the nerve terminal cytoskeleton, which included filamentous linkages among synaptic vesicles. ET revealed novel features of inner membrane for these mitochondria. Crista structure was polarized in that crista junctions, circular openings of the inner membrane under the outer membrane, were aligned with the cytoskeletal superstructure and occurred at higher density in mitochondrial membrane facing the presynaptic membrane. These characteristics represent the first instance where a subcomponent of an organelle is shown to have a specific orientation relative to the polarized structure of a cell. The ratio of cristae to outer membrane surface area is large in these mitochondria relative to other tissues, indicating a high metabolic capacity. These observations suggest general principles for cytoskeletal anchoring of mitochondria in all tissues, reveal potential routes for nonsynaptic communication between presynaptic and postsynaptic partners using this novel cytoskeletal framework, and indicate that crista structure can be specialized for particular functions within cellular microdomains.

Convergence of auditory-nerve fiber projections onto globular bushy cells.

Globular bushy cells are a key element of brainstem circuits that mediate the early stages of sound localization. Many of their physiological properties have been attributed to convergence of inputs from the auditory nerve, many of which are large with complex geometry, but the number of these terminals contacting individual cells has not been measured directly. Herein we report, using cats as the experimental model, that this number ranged greatly (9-69) across a population of 12 cells, but over one-half of the cells (seven of 12) received between 15 and 23 inputs. In addition, we provide the first measurements of cell body surface area, which also varies considerably within this population and is uncorrelated with convergence. For one cell, we were able to document axonal structure over a distance greater than 100 microm, between the soma and the location where the axon expanded to its characteristic large diameter. These data were combined with accumulated physiological information on vesicle release, receptor kinetics and voltage-gated ionic conductances, and incorporated into computational models for four cells that are representative of the structural variation within our sample population. This predictive model reveals that basic physiological features, such as precise first spike latencies and peristimulus time histogram shapes, including primary-like with notch and onset-L, can be generated in these cells without including inhibitory inputs. However, phase-locking is not significantly enhanced over auditory-nerve fibers. These combined anatomical and computational approaches reveal additional parameters, such as active zone density, nerve terminal size, numbers and sources of inhibitory inputs and their activity patterns, that must be determined and incorporated into next-generation models to understand the physiology of globular bushy cells.

Synaptic inputs compete during rapid formation of the calyx of held: A new model system for neural development

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) >200 μm2/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.

Maturation of synaptic partners: functional phenotype and synaptic organization tuned in synchrony

Maturation of principal neurons of the medial nucleus of the trapezoid body (MNTB) was assessed in the context of the developmental organization and activity of their presynaptic afferents, which grow rapidly to form calyces of Held and to establish mono‐innervation between postnatal days (P)2 and 4. MNTB neurons and their inputs were studied from embryonic day (E)17, when the nucleus was first discernable, until P14 after the onset of hearing. Using a novel slice preparation containing portions of the cochlea, cochlear nucleus and MNTB, we determined that synaptic inputs form onto MNTB neurons at E17 and stimulation of the cochlear nucleus can evoke action potentials (APs) and Ca2+ signals. We analysed converging inputs onto individual MNTB neurons and found that competition among inputs was resolved quickly, as a single large input, typically larger than 4 nA, emerged from P3–P4. During calyx growth but before hearing onset, MNTB cells acquired their mature, phasic firing property and quantitative real‐time PCR confirmed a coincident increase in low threshold K+ channel mRNA. These events occurred in concert with an increase in somatic surface area and a 7‐fold increase in the current threshold (30 to >200 pA) required to evoke action potentials, as input resistance (Rin) settled from embryonic values greater than 1 GΩ to approximately 200 MΩ. We postulate that the postsynaptic transition from hyperexcitability to decreased excitability during calyx growth could provide a mechanism to establish the mature 1:1 innervation by selecting the winning calyceal input based on synaptic strength. By comparing biophysical maturation of the postsynaptic cell to alterations in presynaptic organization, we propose that maturation of synaptic partners is coordinated by synaptic activity in a process that is likely to generalize to other neural systems.

Development of gerbil medial superior olive: integration of temporally delayed excitation and inhibition at physiological temperature

The sensitivity of medial superior olive (MSO) neurons to tens of microsecond differences in interaural temporal delay (ITD) derives in part from their membrane electrical characteristics, kinetics and timing of excitatory and inhibitory inputs, and dendrite structure. However, maturation of these physiological and structural characteristics are little studied, especially in relationship to the onset of auditory experience. We showed, using brain slices at physiological temperature, that MSO neurons exhibited sensitivity to simulated temporally delayed (TD) EPSCs (simEPSC), injected through the recording electrode, by the initial phase of hearing onset at P10, and TD sensitivity was reduced by block of low threshold potassium channels. The spike generation mechanism matured between P10 and P16 to support TD sensitivity to adult‐like excitatory stimuli (1–4 ms duration) by P14. IPSP duration was shorter at physiological temperature than reported for lower temperatures, was longer than EPSP duration at young ages, but approached the duration of EPSPs by P16, when hearing thresholds neared maturity. Dendrite branching became less complex over a more restricted time frame between P10 and P12. Because many physiological and structural properties approximated mature values between P14 and P16, we studied temporal integration of simEPSCs and IPSPs at P15. Only a narrow range of relative onset times (< 1 ms) yielded responses showing sensitivity to TD. We propose that shaping of excitatory circuitry to mediate TD sensitivity can begin before airborne sound is detectable, and that inhibitory inputs having suboptimal neural delays may then be pruned by cellular mechanisms activated by sensitivity to ITD.

Molecular Guidance Cues Necessary for Axon Pathfinding from the Ventral Cochlear Nucleus

During development, multiple guidance cues direct the formation of appropriate synaptic connections. Factors that guide developing axons are known for various pathways throughout the mammalian brain; however, signals necessary to establish auditory connections are largely unknown. In the auditory brainstem the neurons whose axons traverse the midline in the ventral acoustic stria (VAS) are primarily located in the ventral cochlear nucleus (VCN) and project bilaterally to the superior olivary complex (SOC). The circumferential trajectory taken by developing VCN axons is similar to that of growing axons of spinal commissural neurons. Therefore, we reasoned that netrin‐DCC and slit‐robo signaling systems function in the guidance of VCN axons. VCN neurons express the transcription factor, mafB, as early as embryonic day (E) 13.5, thereby identifying the embryonic VCN for these studies. VCN axons extend toward the midline as early as E13, with many axons crossing by E14.5. During this time, netrin‐1 and slit‐1 RNAs are expressed at the brainstem midline. Additionally, neurons within the VCN express RNA for DCC, robo‐1, and robo‐2, and axons in the VAS are immunoreactive for DCC. VCN axons do not reach the midline of the brainstem in mice mutant for either the netrin‐1 or DCC gene. VCN axons extend in pups lacking netrin‐1, but most DCC‐mutant samples lack VCN axonal outgrowth. Stereological cell estimates indicate only a modest reduction of VCN neurons in DCC‐mutant mice. Taken together, these data show that a functional netrin‐DCC signaling system is required for establishing proper VCN axonal projections in the auditory brainstem. J. Comp. Neurol. 504:533–549, 2007.