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date: 26 May 2019

Perinatal Development of the Medial Nucleus of the Trapezoid Body

Abstract and Keywords

Bushy cells (BC) of the cochlear nucleus mono-innervate their target neuron, the principal cell of the medial nucleus of the trapezoid body (MNTB), via the calyx of Held (CH) terminal, which is a typically mammalian structure and perhaps the largest nerve terminal in the brain. CH:MNTB innervation has become an attractive model to study neural circuit formation because it forms quickly, passing through stages of competition in mice within 2–4 days. BCs innervate MNTB neurons by E17, but CHs do not begin to grow for another five days (P3). Progress has been made to identify molecular factors for axon guidance, and CH growth, and physiological maturation of synaptic partners, but important details remain to be discovered. We summarize key events in CH formation and highlight unresolved issues in molecular and physiological signaling, roles for non-neural cells, and the nature of competition during the first postnatal week.

Keywords: MNTB, calyx of Held, gene expression, synaptic transmission, synaptogenesis, pruning

The calyx of Held (CH) was first described by Hans Held in 1893, and has since figured prominently in key discoveries about nervous system function. The large size of the CH made it amenable to early study using the light microscope, and its appearance was used as justification for and against the Neuron Doctrine (Ramon y Cajal, 1995; von Gersdorff & Borst, 2002). It is a typically mammalian structure and, despite early controversy (Moore & Moore, 1971; Richter et al., 1983), is now an accepted structure in humans (Kulesza, 2008; Kulesza & Grothe, 2015). The CH is perhaps the largest nerve terminal in the mammalian brain, and has more active zones by species than the cerebellar mossy fiber rosette (CH: 554 in postnatal day (P)9 rat, Satzler et al., 2002; 678 in P14 rat, Taschenberger et al., 2002; 2400 in adult cat, Rowland et al., 2000; mossy fiber rosette: 191–424 in P18 rat, Xu-Friedman & Regehr, 2003; 42–243 in P21 mouse, Kim et al., 2013). The CH, which originates from globular bushy cells (GBC) of the ventral cochlear nucleus (VCN) and forms a 1:1 innervation of principal neurons in the medial nucleus of the trapezoid body (MNTB; Friauf & Ostwald, 1988; Smith et al., 1991; Spirou et al., 1990; Tolbert et al., 1982;), is a key component of binaural convergence in the superior olivary complex (Harrison & Warr, 1962; van Noort, 1969; Warr, 1972). The CH functions at the synaptic level as a reliable and temporally precise relay of neural activity in vivo (Guinan & Li, 1990) with modification of postsynaptic activity likely by inhibitory inputs (Kopp-Scheinpflug et al., 2003, 2008; Awatramani et al., 2004, 2005). The fast and precise excitatory input provided by the CH to MNTB principal neurons translates into system-level function by driving inhibition in binaural circuits, since the postsynaptic MNTB principal cells are glycinergic (Campistron et al., 1986; Finlayson & Caspary, 1989). The main targets of the MNTB are the medial superior olive (MSO), lateral superior olive (LSO) and the superior paraolivary nucleus (SPN), where neuronal activity is based on time of arrival of excitatory and inhibitory inputs (Boudreau & Tsuchitani, 1968; Brand et al., 2002; Felix & Magnusson, 2016; Goldberg & Brown, 1969; Kuwada & Batra, 1999; Yin & Chan, 1990). We and others established the CH:MNTB system as a powerful model for neural circuit formation, due to the rapid pace of transition from small terminal to the readily measured end point of 1:1 innervation onto most neurons in just 3–4 days (Hoffpauir et al., 2006; Holcomb et al., 2013; Kandler & Friauf, 1993). In this review we focus on the timeline for CH:MNTB innervation and highlight important unsolved mysteries.

Embryology

The embryonic origins of the MNTB are poorly understood. MNTB neurons are born in the mouse hindbrain between embryonic day (E)11 and E12 (Figure 1; Pierce, 1973) and the rat hindbrain between E13 and E16 (Kudo et al., 2000). Altman and Bayer (1980) postulated the rhombic lip as the site of MNTB origin, as the rhombic lip is the primary proliferative zone in the hindbrain. However, if this postulate is true, the MNTB must arise from an Atoh1-negative region of the rhombic lip, as the MNTB is not fate-mapped in the Atoh1-Cre lineage (Maricich et al., 2009). The MNTB is derived from progenitor cells in rhombomeres 4 and 5, as it is lineage-labeled with the Hoxb1-Cre (rhombomere 4) and the Egr2-Cre (rhombomeres 3 and 5) transgenic lines (Maricich et al., 2009; Marrs et al., 2013).

Perinatal Development of the Medial Nucleus of the Trapezoid BodyClick to view larger

Figure 1. Temporal correlation of CH and MNTB neuron maturation.

Trapezoid fiber inputs to the MNTB are observed by E17.5. However, there is a waiting period before rapid CH growth begins. Rapid CH growth, measured by an increase in its apposed surface area, occurs between P2–P4 (solid line). During this period, innervation of MNTB neurons by multiple protocalyceal inputs changes to a single large CH. The intrinsic response of MNTB neurons to current injection changes from tonic firing to one or a few spikes at the onset of the current step (phasic firing). Postsynaptic spike threshold also increases during this time, accompanied by an increase in input resistance. By P8, mono-innervation is almost complete and, in parallel, postsynaptic spike thresholds stabilize and intrinsic firing in most MNTB neurons is phasic. Spontaneous activity has been clearly demonstrated in the early postnatal period, but may also be important in establishing brainstem circuitry before E15. Depicted on the left are developmental milestones at different points in the brainstem auditory circuit. Dates of neurogenesis are shown in blue. The age of measurable connectivity between regions is shown with red arrows. Action potential waveforms indicate the first age at which activity was recorded in each region. Refinement of MNTB projections to the LSO occurs from P2 to P8. Expression of genes for myelination can be detected at P4 and myelination of bushy cell axons occurs by P9. PNNs can be detected around MNTB neurons as early as P6 but continue to refine their structure through P14. Most of these developmental milestones occur before the onset of hearing at P10. SGC: spiral ganglion cells, VCN: ventral cochlear nucleus; ASA: apposed surface area.

(Modified from Marrs and Spirou, 2012; Hoffpauir et al., 2010).

The earliest (though not specific) marker for MNTB principal cells is the En1 transcription factor (Altieri et al., 2015; Jalabi et al., 2013; Marrs et al., 2013). Using the En1-Cre strain for lineage tracing, presumptive MNTB neurons can be identified near the ventral edge of the brainstem at E12.5, though they are clustered laterally along with cells that will give rise to other superior olivary nuclei (Altieri et al., 2015; Marrs et al., 2013). The MNTB cells appear to migrate medially from this lateral location, and the nucleus becomes morphologically distinct in its medial position around E17.5 (Marrs et al., 2013). The importance of En1 for MNTB development is highlighted by En1 conditional deletion studies, in which the nucleus forms but is not maintained in postnatal mice (Altieri et al., 2015; Jalabi et al., 2013). In addition, En1 expression is necessary for activation of another MNTB transcription factor, FoxP1 (Altieri et al., 2015; Marrs et al., 2013). A third transcription factor likely to be important for MNTB formation is Ptf1a, which is required for formation of inhibitory interneurons in other neural circuits (Fujiyama et al., 2009; Jusuf et al., 2011). It is unclear if or where Ptf1a fits into the genetic hierarchy of En1 and FoxP1 expression in the developing MNTB.

Although the primary growth period of the calyx occurs postnatally, the initial wiring is established before birth. The VCN is derived from progenitor cells in rhombomeres 2–4 (Farago et al., 2006). Birth of GBC neurons in the VCN peaks at E12 in mouse, whereas the peak birth of the spiral ganglion cells occurs at E11 (Figure 1; Pierce, 1973). GBC axons extend across the midline by E13.5 into the region where the MNTB will form ( Howell et al., 2007; Kandler & Friauf, 1993), though the migration of MNTB neurons is not complete until E17.5 (Marrs et al., 2013). Although MNTB cells are functionally innervated by GBCs as early as E17.5 (Hoffpauir et al., 2010), rapid growth of the calyx does not initiate until P2 after a 4-day waiting period has ensued (Holcomb et al., 2013; Kandler & Friauf, 1993). Whether contacts exist between GBCs and MNTB principal cells prior to E17.5 has yet to be determined, though it is conceivable that the arrival of MNTB neurons through medial migration initiates collateral branching of the ventral acoustic stria axons to form calyceal connections (Kandler & Friauf, 1993).

Axonal projections from the GBC to the MNTB require the netrin-DCC signaling pathway as a midline attractant, as germline mutations in either netrin1 or DCC genes alter axonal outgrowth from the VCN and prevent midline crossing of this contralateral projection (Howell et al., 2007). Likewise, the midline repulsive slit-Robo signaling pathway helps regulate midline crossing. Conditional mutations in Robo3, which are normally permissive for midline crossing, prevent contralateral projection of the ventral acoustic stria and result in aberrant ipsilateral connections when Robo3 is deleted using the Egr2-Cre driver line (Michalski et al., 2013; Renier et al., 2010).

Formation of a Calyx of Held

GBC axons that have arrived within the MNTB during embryonic ages branch profusely and collectively and form 5–20 small contacts on somata of MNTB neurons by P2 (Holcomb et al., 2013). Volume electron microscopy in mice at 1-day intervals, beginning at P2, reveals the emergence of large terminals (>35µm2 of apposed surface area, ASA) onto nearly half the cells by P3, and onto nearly all cells by P4. About 60% of cells receiving large inputs at P3 are contacted by more than one large input, suggesting competition between inputs (Figure 2; panel D). By P6, about 75% of all cells have resolved to mono-innervation via a large CH and, by P9, this number reaches 88% (Holcomb et al., 2013). These two phases of strengthening and pruning are similar to refinement of innervation at the neuromuscular junction (Tapia et al., 2012) except that the time frame at the CH:MNTB is compressed. This short time frame entails rapid assembly of active zones, whereby several hundred are assembled over a time frame of days (Hoffpauir et al., 2006).

Perinatal Development of the Medial Nucleus of the Trapezoid BodyClick to view larger

Figure 2. Growth of the Calyx of Held.

(A) At P2, MNTB principal cell bodies (tan) are relatively smooth, with multiple axons (assorted colors) making contact in an en passant fashion (black arrowheads). No protocalyces are evident at this age. (B) At P3, principal cells have begun to form both short and long projections (white arrowheads). Many inputs have been removed, leaving only a few larger inputs including one solid, cup-like protocalyx (blue) with a few collateral projections (white arrows). (C) By P4, the protocalyx has spread to occupy substantial somatic surface area. Longer collateral projections have formed from both the principal cell (white arrowheads) and protocalyx (white arrows). (D) Similarly sized competing protocalyces (blue, green) occupy distinct territories on the principal cell surface, and both produce collateral projections (white arrows). Collaterals can also be seen from the principal cell (white arrowheads). Scale bars: 5µm.

(Modified from Holcomb et al., 2013, with permission).

As CHs grow, they extend many collateral processes. These collaterals are sometimes tipped with growth cones and the longest collaterals extend >75 µm from the edges of the apposed terminal ( Holcomb et al., 2013; Kandler & Friauf, 1993; Morest, 1968). The presence of growth cone-tipped collaterals and high variability in collateral length could suggest that these collaterals undergo dynamic motility. More recent in vivo two photon experiments performed in perinatal rat pups reported many calyx collaterals exhibit high levels of turnover during one-hour imaging sessions and, further, that these collateral structures can form functional synaptic contacts with off-target principal cells (Rodríguez-Contreras et al., 2008). Collectively, these collateral arbors may aid in the selection of appropriate synaptic partners. Interestingly, significant postsynaptic reorganization parallels the initial growth of the CH. By P2 in rodents, only small terminals are found along the soma of each principal neuron and the neurons are densely packed together within the MNTB (Figure 2, panel A). As protocalyces form over the next two days, the principal cells begin to separate from one another and extend many collateral structures, termed somatic spicules (Figure 2; Holcomb et al., 2013). The function and dynamics of these structures has yet to be explored.

Each principal neuron in the MNTB is surrounded by specialized lattice-like structures of extracellular matrix, known as perineuronal nets (PNNs: see chapter by Morawski and colleagues for greater detail). PNNs begin forming during the first postnatal week (Blosa et al., 2013; Kolson et al., 2016). They are composed mostly of chondroitin sulfate proteoglycans (such as aggrecan and brevican), hyaluronan, Hapln linker proteins, and tenascin-R (reviewed by Wang & Fawcett, 2012). Their exact functions are still being investigated, but they are involved in the control of synaptic plasticity and regulation of synaptic activity (Balmer, 2016; Carulli et al., 2010; reviewed by Sorg et al., 2016). In the visual cortex, Hapln1 knockout mice demonstrate a functional gain in the ability to compensate for loss of connectivity following denervation (Carulli et al., 2010). This study suggests that the formation of PNNs serves to limit synaptic plasticity after a critical period. Thus, it is intriguing to think that PNNs in the MNTB may cement and stabilize the large CH terminal and inhibit multi-innervation at more mature ages. Microarray analysis detects significantly changing transcript levels for three main PNN components (aggrecan, brevican and Hapln1), all of which increase between P0 and P6 when the CH is growing and refining to mono-innervation (Kolson et al., 2016). Immunofluorescence shows the linker protein, Hapln1, is visible as early as P0 in the MNTB. However, there is a delay between protein expression of the linker protein and the other PNN components because brevican and aggrecan are not detectable until after P6 (Figure 1; Kolson et al., 2016). Brevican knockout mice show a delay in synaptic transmission between the CH and principal neurons along with prolonging of action potentials when compared to wildtype controls at P23–31, but these mice have not been evaluated at early developmental ages (Blosa et al., 2015). Although the overall structure of the PNNs is comparable between the brevican knockout and wildtype mice (Blosa et al., 2015), other components of the PNNs, such as aggrecan, still are present in the knockouts and may suggest redundant function. However, more extensive perturbations, such as double and triple knockouts, or knockout of the linker protein, Hapln1, and their effects on CH refinement to mono-innervation remain to be investigated.

Cell Signaling in MNTB Development

The CH terminal grows to its large size in synchrony with biophysical maturation of the principal neuron and an increase in principal neuron size (Hoffpauir et al., 2006, 2010; Rusu & Borst, 2011; see later section in this review). The contralateral CH projection to principal neurons is also tonotopically organized (reviewed by Kandler et al., 2009). Notably, MNTB innervation of LSO transitions from a relatively broad territory along the tonotopic axis at P2 to a narrowed territory by P8 (Figure 1; Kim & Kandler, 2003), suggesting that the MNTB-LSO connection sharpens in parallel with maturation of the CH and principal neuron. This coordinated maturation of pre- and postsynaptic partners and functional refinement of synaptic projections to LSO suggests mechanisms of cell signaling to regulate maturation of the systems. Genetic perturbation studies are a useful means of testing the functionality of such signaling interactions. This section provides a review of genetic perturbations that affect early development of either the GBC or the principal neuron and their effects on CH-principal neuron connectivity, morphology and size.

The Fmr1 gene is associated with fragile X syndrome (FXS) and autism spectrum disorder (ASD; Hagerman et al., 2010). Interestingly, patients with FXS and ASD present with abnormal auditory brainstem responses (ABRs; Roth et al., 2012). Fmr1 knockouts have been used to model these disorders and examine effects in the MNTB (Rotschafer et al., 2015; Ruby et al., 2015). Compared to their wildtype counterparts, the ABRs of Fmr1-/- mice have elevated thresholds and smaller amplitudes of waves I and III, indicating both peripheral and central deficits. Neuronal cell size is reduced in both bushy cells and principal neurons, and there is an increase in the number of VGAT+ punctae in the MNTB, suggesting an altered excitatory:inhibitory synaptic balance (Rotschafer et al., 2015). A similar phenotype is observed in Fmr1-/- rats, which have smaller principal neurons and fewer calyces (Ruby et al., 2015). The protein product of the Fmr1 gene, FMRP (Fragile X mental retardation) negatively regulates mRNA translation and binds Kv3.1b mRNA in the brainstem (Strumbos et al., 2010). Kv3.1b is normally expressed along a tonotopic gradient in MNTB, with the highest expression in the medial MNTB (Li et al., 2001). Fmr1-/- mice have disrupted Kv3.1b tonotopy and do not show the increase in Kv3.1b expression following high-frequency stimulation that is found in controls (Strumbos et al., 2010). An intriguing hypothesis is that the disruption of ion channel distribution along the tonotopic gradient affects neural activity, which in turn may be related to the decrease in cell size. In wild-type mice, the lateral low-frequency encoding neurons are larger than their medial counterparts (Weatherstone et al., 2017). Similar to Fmr1-/- rodents, in Deafwaddler mice, which have a spontaneous mutation in the Pmca2 (plasma membrane calcium ATPase 2) gene, the neuronal size gradient is disrupted across the tonotopic axis. It is noteworthy that the phenotype of decreased cell size is also observed in human patients with ASD (Lukose et al., 2015).

Many signaling pathways regulate different steps in CH formation, including where a calyx projects (ipsilateral vs. contralateral), how large and complex the terminal grows, and the subsequent refinement to mono-innervation. The GBC normally projects its axon to form the CH in the contralateral MNTB (Cant & Casseday, 1986). In addition to the netrin-DCC and slit-Robo signaling pathways mentioned earlier, an important signaling component mediating this projection pattern is the interaction of the Eph receptor tyrosine kinase family with Ephrin ligands. The Eph signaling pathway is unique in that both traditional forward signaling, initiated from the ligand and processed through the receptor, and reverse signaling, which involves signaling initiated by the extracellular domain of the receptor and processed through the membrane-bound ligand, can occur. In a series of knockout and dominant negative overexpression experiments, a 10% increase in ipsilateral calyx formation was seen in EphB2/EphB3 double knockout mice. Furthermore, reverse signaling through EphB2-EphrinB2 promotes contralateral calyx formation and inhibits ipsilateral sprouting, because ephrin-B2lacZ mice, where the cytoplasmic domain of Ephrin-B2 is replaced by β-galactosidase, exhibited a significant increase in ipsilateral calyx formation (Hsieh et al., 2010). EphrinB2 is not expressed in the principal neurons until after E17 when the initial synaptogenesis has already occurred (Hsieh et al., 2010; Marrs & Spirou, 2012) and therefore does not seem to be acting as a typical guidance factor. Rather, EphrinB2 seems to act as a repulsive signal to inhibit ipsilateral sprouting of an axon that has already crossed the midline, possibly interacting with developing PNNs to limit ipsilateral connections postnatally (Blosa et al., 2013; Kolson et al., 2016).

The contactin superfamily consists of a group of cell adhesion molecules whose expression is enriched in the developing nervous system (reviewed by Shimoda & Watanabe, 2009). Expression of Cntn5 (contactin 5) is important for mediating adhesion of the calyceal contact with the principal neuron rather than a role in axon migration because the mRNA is not expressed until after P1, when initial synaptogenesis has already occurred. Whereas nearly all principal neurons are contacted by a large CH terminal at P6 in wildtype mice (Holcomb et al., 2013; Toyoshima et al., 2009), 8% of principal neurons lack a CH terminal in Cntn5-/- mice. The neurons that lack calyceal innervation later undergo apoptosis due to lack of innervation (Toyoshima et al., 2009).

Several signaling pathways affect calyx size, morphology and refinement to mono-innervation, but most perturbations yield an incomplete and often mild phenotype, suggesting that formation of the large CH terminals is a robust process. The Sad-A and Sad-B kinases are a pair of serine/threonine kinases that localize within the CH terminal. Double conditional knockout mice, where Cre expression is driven by the Parvalbumin promoter, which is expressed both presynaptically at birth and postsynaptically by the end of the first postnatal week, have significantly smaller CH terminals when evaluated at P24 (Lilley et al., 2014). Genetic conditional deletion of two receptors for Bmp signaling, Bmpr1a/b, results in smaller terminal size and an almost 40% reduction in the number of mono-innervated principal neurons at P7–10 (Xiao et al., 2013). Electrophysiological studies show that the timing of synaptic transmission is impaired in the Bmpr1 knockout mice. Smaller excitatory postsynaptic currents (EPSCs) in the knockout mice correspond with delays in the rise time of the excitatory postsynaptic potentials (EPSPs) and action potentials, which occur with long delays. However, the first phenotypic differences cannot be observed until P3, suggesting that initial synaptogenesis of small glutamatergic inputs is not altered in these knockout mice (Xiao et al., 2013). These data suggest that Bmp signaling is required for the initial stage of growth of large terminals and that further growth and competition are inter-related processes that establish mono-innervation.

Further evidence supporting the idea that fast neurotransmission drives CH growth is demonstrated through Egr2-driven conditional knockout of the dynamin family of GTPases that are required for clathrin-mediated endocytosis, Dnm1/3 (Fan et al., 2016). Similar to the Bmp conditional knockout, initial synaptogenesis is not affected in the Dnm1/3 conditional knockout mice. Wild-type mice demonstrate a significant functional maturation in the kinetics of spontaneous EPSCs (sEPSCs) across development, but the knockout mice do not display this developmental transformation in fast firing properties, and sEPSCs have slower rise and decay times. Additionally, the principal neurons in the knockout mice display more immature biophysical properties, including a higher membrane excitability and frequency of spontaneous action potentials. However, in stark contrast to most of the previously listed genetic perturbations, the Dnm1/3 conditional knockout mice have a severe phenotype, with a lack of initial growth of terminals into the protocalyx phase and accumulating degeneration of MNTB neurons (Fan et al., 2016). The drastic phenotypes seen in this knockout model may be due to deficits in the balance of exo- and endocytosis, and suggest that a minimal level of endocytosis is required for CH growth. Alternatively, generalized effects on cell signaling could account for this extreme phenotype. It is worth noting that in both the Bmpr and Dyn1/3 cKO mice, both presynaptic and postsynaptic gene expression was affected, so that altered function in both synaptic partners could underlie alterations in CH maturation.

Functional Maturation of Synaptic Partners

Functional synaptic input to the MNTB from VCN begins during embryonic ages. By E15.5 in mice, both auditory nerve and VCN neurons can generate action potentials (AP), and by E16.5, APs can be reliably evoked in VCN neurons by auditory nerve fiber stimulation. At E17.5, the earliest age at which the MNTB is recognizable in tissue sections, a single stimulus pulse initiates a series of subthreshold inputs of varying latency that can generate spikes in the MNTB principal cell (Hoffpauir et al., 2010; Marrs & Spirou, 2012). In response to direct activation of VCN inputs to the MNTB, synaptic latencies shorten during the first postnatal week, postsynaptic spikes increase in height and decrease in duration, and tonic firing changes to an adult-like phasic response (Figure 3; Hoffpauir et al., 2010; Sierksma et al., 2017).

Perinatal Development of the Medial Nucleus of the Trapezoid BodyClick to view larger

Figure 3. Physiological responses in the MNTB during embryonic and early postnatal ages.

(A, B) Activation of the contralateral MNTB by auditory nerve stimulation at E17. (A) Whole-head mouse slice illustrating the auditory brainstem circuit from the spiral ganglion to the MNTB. The auditory nerve is stimulated at the base of the cochlea and recordings are made from principal cells in the contralateral MNTB. SG: spiral ganglion; VCN: ventral cochlear nucleus; ipsi: ipsilateral; contra: contralateral. (B) Responses in an MNTB principal cell at E17 evoked by auditory nerve stimulation. Top trace: synaptic currents recorded under voltage-clamp to a single shock delivered to the auditory nerve. Bottom trace: Changes in membrane potential recorded under current clamp to the same auditory nerve stimulus. Synaptic currents show multiple components. An initial rapid current of ~150 pA is followed by several smaller currents (~50 pA), suggesting multiple presynaptic inputs on the principal cell. The principal cell responds to synaptic input with a delayed spike that is evoked by EPSP summation. (C) MNTB principal neurons transition from tonic to phasic firing during the period of rapid CH growth. Intrinsic firing to somatic current injection is illustrated at P2 and P4. At P2, depolarizing current steps evoke sustained firing while, at P4, phasic firing occurs at the onset of the current pulse at all depolarizations. Hyperpolarizing currents evoke a sag in membrane potential at P2 that continues to occur at P4. Current steps are 200 ms long.

(Modified from Hoffpauir et al., 2010; Marrs and Spirou, 2012).

Spontaneous Activity during the Period of CH Growth

Since formation of the CH occurs before the ear canals open, this process is considered to be independent of environmental sound, given the caveat that pups may self-stimulate their cochlea by vocalization. Spontaneous activity has been demonstrated in the MNTB, but its role in CH formation is debatable. Blockade of inner hair cell activation and synaptic transmission by genetic deletion of Cacna1d (Cav1.3) yields a smaller MNTB with 1/3 fewer neurons (and similar changes across the superior olivary complex; Hirtz et al., 2011) but with, at best, subtle effects on CH formation and neurotransmission onto the remaining neurons (Erazo-Fisher et al., 2007). Slc17a8 (vesicular glutamate transporter 3) knockout does not alter the biophysical properties of MNTB neurons or their excitability by glutamate photo-uncaging and, similar to otoferlin knockout, does not alter innervation of LSO by MNTB (Noh et al., 2010), suggesting little effect of cochlear silencing on CH formation. Likewise, CHs form and mature normally by P20 in dn/dn mice, which lack auditory nerve spontaneous activity (Leao et al., 2006b; Youssoufian et al., 2005). However, these mice exhibit reduced but present spontaneous activity, thus GBCs may generate activity in the absence of cochlear input.

Spiral ganglion neurons are spontaneously active in vitro at embryonic ages (earliest age tested, E14), (Marrs and Spirou, 2012), suggesting that synaptic connections, functional from auditory nerve to MNTB by E17.5, permit a flow of spontaneous activity through these neural circuits beginning in the embryo. Starting at about P2, characteristic bursts (~10 Hz) and minibursts (~100 Hz) of spontaneous activity, which are likely of cochlear origin, can be recorded from GBC axons and MNTB neurons (Clause et al., 2014; Sierksma et al., 2017; Tritsch et al., 2007; Tritsch & Bergles, 2010). Simulations of the contributions of intrinsic and synaptic conductances to burst firing suggest that activation of NMDA receptors on principal neurons increases both the number of spikes as well as spike frequencies within minibursts, but not bursts, relative to GBC input (Sierksma et al., 2017). NMDA receptors are predicted to have a stronger effect on bursting at P3 than at P5 or P6 (Sierksma et al., 2017), which agrees well with the measured decline in the NMDA contribution to EPSCs during the early postnatal period, and the shift in balance towards a more rapidly decaying AMPA-centric synaptic response (Hoffpauir et al., 2010; Joshi & Wang, 2002). Taken together, the early postnatal presence of bursting spontaneous activity in the MNTB and the embryonic presence of spontaneous spiking in spiral ganglion cells suggest that early spontaneous activity in the pathway between the spiral ganglion and MNTB may create a state of activity-dependent homeostatic regulation of CH:MNTB innervation. Clause et al. (2014) demonstrated that altering spontaneous firing in the spiral ganglion does not affect the average rate of spontaneous firing in the MNTB, but changes its temporal pattern, without effect on CH morphology. Patterned spontaneous activity shapes neuronal maps by pruning extensive branching of afferent axons (Shatz, 1996). The formation of functional pre-calyx contacts onto MNTB neurons in the Dnm1/3 knockout does not preclude roles for spontaneous activity in this step of maturation, especially if vesicle fusion rates are low (Fan et al., 2016). The possibility remains, then, that spontaneous activity plays roles in maintaining bouton-like synaptic contacts measured from E17.5, transition to the protocalyx stage, and resolving competition among supernumerary inputs.

NMDA-dependent bursting may strengthen activity in the immature CH:MNTB synapse through activation of small conductance apamin-sensitive, Ca++-dependent K+ channels (SK channels). SK channels are activated by bursts of spikes in MNTB neurons that produce calcium sparks, and by calcium influx through NMDA receptors. They give rise to spontaneous outward currents that regulate the resting membrane conductance and the resting potential of MNTB neurons. The number of MNTB neurons that exhibit calcium sparks and SK-dependent transient outward currents gradually increases from P6 (40%) to hearing onset around P10 (~90%), and then decreases by P14 (5%), suggesting that this temporary feature is pertinent to synapse maturation (Zhang & Huang, 2017) and perhaps the initial stages of CH formation. Embryonic and early postnatal spontaneous activity may therefore regulate ion channel expression through NMDA receptor activation in the developing MNTB well before sound-induced cochlear drive becomes established. In addition to spontaneously active VCN inputs, MNTB neurons at embryonic and early postnatal ages also receive tonic and phasic depolarizing GABAergic and glycinergic inputs that regulate membrane excitability (Hoffpauir et al., 2010; Awatramani et al., 2005).

Changes in Postsynaptic Excitability during CH Growth

CH growth and presynaptic pruning are accompanied by a reduction in postsynaptic excitability. Small currents of ~30 pA can evoke APs at E17.5–P2, but these values rapidly increase nearly tenfold by P4 (Hoffpauir et al., 2010). During this time frame, CHs grow from bouton size into cup-shaped terminals up to 400 µm2 in ASA with the postsynaptic cell (Holcomb et al., 2013). By P4, postsynaptic membrane resistance decreases and low-threshold K+ subunits, hyperpolarization-activated cation channels and leak channels are upregulated ( Berntson & Walmsley, 2008; Hoffpauir et al., 2010; Leao et al., 2005a; Leao et al., 2005b; Leao et al., 2006a; Sierksma & Borst, 2017; Sierksma et al., 2017). Consequently, sustained spiking during current injection changes to an adult-like phasic response (Figure 3). Developmental increases in the peak Na+ current and its faster inactivation, and increases in the magnitude of delayed rectifier K+ currents (Elezgarai et al., 2003; Ishikawa et al., 2003; Leao et al., 2005a) are confirmed by upregulation of Kcna gene family expression (Kcna2, Kcnab3) and of the Scn1a gene expression between P0-P6 (Kolson et al., 2016).

Increases in EPSC amplitude occur along with changes in postsynaptic input resistance. Between P3-P6, EPSC amplitudes gradually increase from the pA to nA range (Hoffpauir et al., 2010; Iwasaki & Takahashi, 1998). Using minimal stimulation of the trapezoid fiber bundle in slices containing the MNTB, Hoffpauir et al. (2010) showed that ~50% of principal neurons between E18 and P0 are driven by small inputs. At P1, this percentage decreases to 33% and by P3, there are no principal neurons driven by small inputs. Even at more mature CH:MNTB synapses (P16–19), spike failure in the MNTB during repeated stimulation occurs when EPSCs become smaller than ~300 pA (Grande & Wang, 2011). Thus, at these early ages, MNTB neurons are unable to follow repeated input. Both short-term synaptic facilitation and depression are present to varying degrees at early postnatal ages and modulate the ability of MNTB neurons to follow trapezoid fiber stimulus rates (reviewed by Borst & van Hoeve, 2012). Nonetheless, the ability of the postsynaptic response to follow CH input increases as the synapse matures towards hearing onset, with the principal neuron achieving a 90% success rate in following trapezoid fiber stimulation by the end of the second postnatal week (Wu & Kelly, 1991).

The improved ability of the MNTB neuron to follow input is also illustrated by a decrease in its input resistance during the embryonic and early postnatal period. Input resistances, which are as high as 1 GΩ at E17, decrease to ~200 MΩ by P6, after which they do not change. Age-dependent decreases in postsynaptic excitability are also illustrated by the finding that, at E17, the maximum slope resistance occurs at membrane potentials more hyperpolarized than the resting membrane potential, whereas by P6, maximum slope resistances are observed at resting potentials (Hoffpauir et al., 2010). Pre- and postsynaptic changes during early development therefore appear to be remarkably well-orchestrated.

Regulation of Calcium during CH Growth

As a general feature of synaptic development, correlated pre-and postsynaptic maturation and the refinement of developing synaptic connections by spontaneous activity are strongly linked to calcium signaling. The temporal dynamics and mode of calcium entry into developing nerve terminals and their postsynaptic counterparts confer specificity to downstream events such as synaptic pruning and dendritic arborization (reviewed by Rosenberg & Spitzer, 2011), and are critical to the rate of outgrowth of the presynaptic growth cone (Gomez & Spitzer, 1999; Vonhoff & Keshishian, 2017). The spatiotemporal calcium profile in the developing CH is therefore likely to be a crucial factor in the early establishment of mono-innervation in the MNTB.

The class of presynaptic Ca++ channels triggering transmitter release from the CH switches early in postnatal development, suggesting spatiotemporal changes in the presynaptic calcium profile during and immediately following establishment of the 1:1 CH:MNTB connection. At P6, a third of the presynaptic Ca++ current flows through high-threshold N-type Ca++ channels sensitive to ωCgTx GVIA, and the P/Q type Ca++ channel blocker ωAga-IVA blocks most of the remaining current. By P8, the N-type Ca++ channel contributes to only 10%, and the P/Q channel to most, of the remaining presynaptic Ca++ current (Iwasaki et al., 2000; Iwasaki & Takahashi, 1998). There is a general decrease in expression of Ca++ channel genes in the MNTB during this time (Cacna1b, Cacna1e, Cacna1g, Cacnb3; Kolson et al., 2016), which may indicate an overall refinement of calcium channel subtypes in principal neurons (Bollmann et al., 1998) and other cells, including glia. Given the likely importance of early presynaptic calcium profiles in establishing monoinnervation in the MNTB, presynaptic Ca++ currents at ages younger than P4, which have not been measured, will provide valuable information about developmental changes in CH Ca++ channel subtypes.

The shift from multiple Ca++ channels to a single class of channels in the CH accompanies a shortening of the distance between Ca++ channels and Ca++ sensors and the formation of Ca++ nanodomains (Wang et al., 2008; Kochubey et al., 2009). Nanodomain formation, which is mediated by septin 5 and SNARE proteins (Yang et al., 2010), preserves temporal precision across the synapse (Yang et al., 2014). The finding that Ca++ sensor sensitivity is lower at P9 than at P16 (KD values of ~80 μM and 120 μM, respectively; Kochubey et al., 2009; Wang et al., 2008) suggests, by extrapolation, that Ca++ channels are not tightly coupled to Ca++-sensors during the first postnatal week, but this has yet to be determined. The slow Ca++ buffer EGTA is more effective at attenuating transmitter release at P8 than at P16, and Ca++ cooperativity values are higher in younger CHs (Fedchyshyn & Wang, 2005). This finding supports poorer spatial coupling between Ca++ channels and Ca++ sensors, as well as the requirement for a higher number of Ca++ channels to trigger transmitter release during early development. Computer simulations demonstrate that tight Ca++ channel-Ca++ sensor coupling almost doubles the Ca++ concentration seen by the Ca++ sensor (Naraghi & Neher, 1997; Schneggenburger & Neher, 2000). Thus, as the synapse matures during the second postnatal week, fewer Ca++ channels are needed to trigger transmitter release and endogenous Ca++ buffering is less likely to saturate with high-frequency stimulation. The early establishment of Ca++ nanodomains might therefore factor into the development of the low C-affinity of the transmitter release process at this synapse.

Fast endogenous calcium buffering in CH terminals becomes more prevalent with age and appears to be specific to the type of calcium binding protein. Between P3 and P6, parvalbumin forms the primary calcium buffer, and is present in almost all CHs as measured by immunohistochemistry (Felmy & Schneggenburger, 2004; Kolson et al., 2016). This fraction remains constant with further development (Felmy & Schneggenburger, 2004), suggesting that presynaptic calcium buffering is established as CHs complete their phase of most rapid growth. In contrast, calretinin is expressed in very few CHs at P6 (~5%) and in an only slightly larger fraction after hearing onset (P14; 18%). Furthermore, calretinin content appears to be highly variable in the CH population at P6, with calretinin-negative and -positive CHs interspersed (Felmy & Schneggenburger, 2004). Since presynaptic calcium transients at P6 decay much slower than those at P11 (2.5 s and 0.9 s, respectively; Chuhma & Ohmori, 2001), calcium extrusion rates are critical at early postnatal ages. The slow rate of calcium buffering by parvalbumin makes it effective on intracellular calcium transients after an action potential, with a primary influence on short-term plasticity (Caillard et al., 2000). Calretinin, in contrast, with a fast buffering capacity similar to that of BAPTA, regulates fast transmission evoked by single spikes (Schwaller, 2014). Its buffering is also more highly activity-dependent than that of parvalbumin (Schwaller, 2010). Thus differences in the prevalence of parvalbumin and calretinin in young CHs, and the heterogenous distribution of calretinin, suggest that co-localization of presynaptic fast and slow calcium buffers would differentially affect calcium extrusion rates in the CH population and may have a proportionally larger role in CH:MNTB development during the first postnatal week. Interestingly, decreases in noradrenaline sensitivity of presynaptic calcium currents point to a developmental shift in the regulation of presynaptic calcium channels by inhibitory G-protein pathways (Leao & von Gersdorff, 2002).

Glia in MNTB Development

Neuroglia in the MNTB, as in other brain regions, serve a diverse and important array of functions. As in the cerebral cortex (Parnavelas, 1999) and cerebellum (Buffo & Rossi, 2013), glial cells in the brainstem are believed to be derived from the ventricular neuroepithelium, from which radial glial processes project to create a “scaffold” that may be the basis for later neural organization (van Hartesveldt et al., 1986; Gomez et al., 1990). Both astrocytes and oligodendrocytes are present in the MNTB as early as P0 in mice and rats, with microglia appearing in substantial numbers only after P6 (Dinh et al., 2014; Saliu et al., 2014). Using EdU injections of pregnant rat dams, Saliu et al (2014) showed that glial progenitor cells in the rat MNTB begin to divide prenatally (E19 or earlier), rapidly increase their rate of division until around the onset of hearing (approximately P12), and then slow their division exponentially until barely distinguishable at P18. EdU-labeled cells later stain positive for the commonly used astrocyte marker, S100β. This timeline of glial proliferation occurs in synchrony with the developmental timeline of the CH terminal (Saliu et al., 2014). Although S100ß is often used as a marker for astrocytes, this protein is also expressed in NG2+ oligodendrocyte precursor cells (OPCs; Hachem et al., 2005). Therefore, proliferating glia in the MNTB may potentially assume either an oligodendrocytic or astrocytic fate (Buffo & Rossi, 2013; Hamori et al., 1997).

Both astrocytes and OPCs participate in a multi-partite synaptic system in the MNTB that is only partially understood. Light and ultrastructural analyses of the developing MNTB have shown that vellous processes, either from astrocytes or OPCs, occupy non-innervated territory on the principal cell surface, surround growing terminals, and interpose between the principal cell and terminals (Dinh et al., 2014; Elezgarai et al., 2001; Holcomb et al., 2013; Reyes-Haro et al., 2010). Dinh et al. (2014) showed that vellous processes, present as early as P0, express S100ß, aldehyde dehydrogenase 1 family member L1 (ALDH1L1), or glial fibrillary acidic protein (GFAP), though the pattern of expression differs for each protein. From P0 to at least P23, ALDH1L1+ glial processes associate with MNTB principal neurons and, later in development, the growing calyx. Processes expressing S100ß are similarly associated with these structures, though the processes were found to be more homogenously distributed throughout the MNTB. GFAP shows no expression within the MNTB at P0 and expression only in glial processes within the MNTB from P6 to P24. No GFAP+ cell bodies are found present in the MNTB at any age studied (Dinh et al., 2014). However, the extent of overlap in expression of these proteins in these processes has yet to be determined. A single astrocyte or OPC may also contact multiple principal cells, opening the possibility for regulation of network-level organization by these cells. In some cases, vellous processes can completely block processes filled with vesicles (presumably calyceal projections) near the principal cell surface from establishing contact, suggesting they may play a direct physical role in competition during calyx development (Holcomb et al., 2013). Additionally, these vellous processes contain metabotropic glutamate receptors (mGluR2/3, P0-24 rats, P6–18 mice) and transporters (EAAT1[GLAST], EAAT2[GLT-1], P10–15 rats), allowing them to respond to glutamate release from the CH, take up excess glutamate, and prevent glutamate receptor saturation in the immature calyx (Elezgarai et al., 2001; Renden et al., 2005; Uwechue et al., 2012). As the calyx matures and fenestrates (P16 and later), the risk of receptor saturation decreases, glutamate uptake by glial cells is less necessary, and expression of mGluR2/3 decreases in these processes (Elezgarai et al., 2001; Renden et al., 2005). Astrocytes may also influence synaptic refinement through gliotransmission and the induction of neuronal slow inward currents (nSICs); repeated stimulation of midline axons causes astrocytes to release both glutamate and D-serine that, together, co-activate NMDA receptors on the principal cell and induce nSICs (Reyes-Haro et al., 2010). In contrast, direct, AMPA receptor-mediated synaptic connections are established between the CHs and OPCs, allowing these cells to receive synchronized input (Müller et al., 2009). Glutamatergic input to NG2+ OPCs has generally been shown to inhibit differentiation of these cells into more mature oligodendrocytes (Gallo et al., 1996 Karadottir & Attwell, 2007; Yuan et al., 1998). However, it has been demonstrated more recently in the rat corpus callosum that the frequency of stimulation has a direct influence on OPC proliferation and differentiation, with 5 Hz stimulation increasing OPC differentiation and 25 Hz and 300 Hz stimulation increasing OPC proliferation (Nagy et al., 2017). As activity can influence both GBC axon diameter and myelin thickness (Sinclair et al., 2017), it is intriguing to think that changes in firing patterns may allow terminals to regulate their own oligodendrocyte differentiation and, therefore, their own axon myelination. The studies of both astrocyte gliotransmission and CH:OPC synapses were performed in 8–10 day old mice, after monoinnervation has already been established. Future studies will need to focus on earlier postnatal time points to elucidate the reciprocal effects that neuron-glial interactions may have on development of the MNTB.

Myelination

Wrapping of axons by myelin sheaths provides an electrically insulating layer that allows the rapid and temporally precise propagation of action potentials. Functionally, the integration of binaural signals and regulation of conduction timing is controlled through the morphological characteristics of axon diameter, myelin thickness, node formation and localization of ion channels (Ford et al., 2015, Seidl & Rubel, 2016; Xu et al., 2017). Myelination in the MNTB, as detectable by immunohistochemistry, begins by P9, when most MNTB principal neurons are monoinnervated by CHs (Saliu et al., 2014). A noticeable increase in myelination is evident around the time of ear canal opening at P10–12, with the developmental growth of axon diameter, increase in myelin thickness, and associated conduction velocity, increasing until P25 and P35, respectively (Sinclair et al., 2017). Myelin wraps leave axon membrane exposed at nodes of Ranvier and at heminodes leading into the nerve terminal. Nav1.6, Navβ4, and Kv3.1 channels are concentrated at the heminode leading into the CH, with fewer channels in the nerve terminal proper (Berret et al., 2016; Leao et al., 2005a). Nav channel clusters at the CH axon heminode in rats are not detectable by IHC at P6. Broad expression of Nav channels begins at P8, and complete clustering at the heminode occurs by P16 (Xu et al., 2017). Nav1.2-mediated Na+ currents and the expression of functional AMPA receptors allow a subpopulation of excitable, immature pre-myelinating oligodendrocytes to generate action potentials in response to current injections. Following knockdown of Nav1.2 channels in immature oligodendrocytes, the expression of myelin basic protein decreases, suggesting a functional role for excitable oligodendrocytes in forming compact myelin (Berret et al., 2017). Computer simulations utilizing morphological measurements of internode length, internode axon diameter, and node diameter predict that the differences associated with tonotopic arrangement of the MNTB adjust the conduction velocity and timing of action potentials for sound localization (Ford et al., 2015).

Specifying CH Competition and Growth

Despite much progress in specifying features and factors in CH growth and MNTB maturation in general, a close view of the earliest steps and mechanisms for these events remains to be worked out. For example, a common theme in neural development is exuberant innervation of a postsynaptic territory, followed by selective strengthening of some inputs and pruning of others. Perhaps the best studied example is the neuromuscular junction (NMJ), which undergoes two phases of strengthening and pruning. At birth, motor end plates are innervated by 10 to 20 small inputs (Tapia et al., 2012). Two or three of these inputs enlarge as the others are pruned. These remaining inputs vie for territory on the motor end plate through a process that can extend for longer than one week. This latter process defines a second phase of strengthening and pruning that resolves to mono-innervation (Balice-Gordon & Lichtman, 1993; Walsh & Lichtman, 2003).

The CH innervation of MNTB neurons in adults resembles the NMJ in that multiple (up to about 20) small inputs innervate the MNTB cell body prior to CH growth, and a single CH mono-innervates its target neuron in adults. However, there is not yet a clear picture of the transition from multiple small inputs to mono-innervation. This lack of clarity is due in part to the speed of CH growth, which was estimated to be, at minimum, the addition of 200 µm2 of ASA per day (Holcomb et al., 2013). Kandler and Friauf (1993), in their classical study using tract-tracing techniques, did not find a single instance of dual innervation of an MNTB neuron by growing CHs. More recently, in vivo juxtacellular and whole cell electrophysiological recording of MNTB neurons shows evidence for dual innervation in at most 3/132 cells (2%) at P3 and P4 (Sierksma et al., 2017). In contrast, minimal stimulation in vitro yields a higher percentage of multiply innervated cells (4/29 (13%) at P4; Hoffpauir et al., 2006) and at P7–12 (6/101 (6%); Bergsman et al., 2004). Since in vitro recordings represent an underestimate due to sectioning of many fibers at the tissue surface near the recording, the results from Bergsman et al. (2004) in older animals imply that a small percentage of MNTB neurons may be multiply innervated around hearing onset.

As a more objective means of assessing multiple inputs, Holcomb et al. (2013) employed volume electron microscopy across a unique time series of images, covering daily intervals at P2–4. Animals were carefully timed at one half day resolution relative to birth. At P2, large diameter fibers branch profusely, yielding 5–20 small, but no large, inputs onto MNTB cell bodies. Just 24 hours later, 43% (16/37) of MNTB cells are contacted by large terminals, and over one-half of these (56%, 9/16) are contacted by two, and in one instance three large nerve terminals. These data from P3 are instructive since they capture the CH growth process on its first day, and they indicate a large percentage of MNTB neurons with competing inputs (Figure 3). Knockout of Bmp1 receptors delays CH growth and a majority (~3/4) of MNTB cells are multi-innervated when tested at P14-16 (Xiao et al., 2013). The most parsimonious interpretation is that competition failed to resolve onto these neurons, although supernumerary innervation could have been induced by the genetic manipulation. Resolution of these EM images, in vitro physiology and genetic manipulation data with other experiments may lie in the fact that competition seems to occur when inputs are still early in their growth phase (Hoffpauir et al., 2006; Holcomb et al., 2013). Most competing inputs at P3 and P4 in these studies were less than 100 µm2 (ASA), and more than one-half of these were less than 50 µm2. Both inputs onto the same cell exceeded 100 µm2 in only one case at each of P3 and P4. These smaller inputs may have eluded detection as contacting the same cell in light microscopy studies, or had too small a physiological signature to be differentiated, especially given the unreliability of linking large postsynaptic currents in vivo to prepotentials. Small inputs also may exhibit less spontaneous activity and be more difficult to detect (Sierksma et al., 2017). Furthermore, volume EM shows that, beyond a size threshold at each age older than P3, the largest inputs all mono-innervate their targets (Hoffpauir et al., 2006; Holcomb et al., 2013).

Summary

In recent years, studies of neural development have revealed new roles for both neurons and glia in formation of neural circuits. In the MNTB, the route from multiple small inputs to monoinnervation is faster than in other mammalian systems, achieving >80% completion within four days. Associated with this transition are birth and death of glia, pruning of inputs, growth and rearrangement of principal MNTB neurons and refinement of their projections to the LSO. Partially overlapping, but largely following from these events, are other steps in maturation such as completion of the formation of perineuronal nets and myelination of CH axons. An overall picture of tissue transformation emerges with likely undiscovered types of cellular and molecular communication among all constituent cell types. The coming years will be exciting as these mechanisms are revealed at a systems level of action and interaction.

Acknowledgments

This work was supported by NIH grants R01 DC007695 (GS), F31 DC014393 (PH) and CoBRE GM103503.

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