Activity-dependent formation and location of voltage-gated sodium channel clusters at a CNS nerve terminal during postnatal development

Clustering of voltage-gated sodium (Na_v) channels and their distribution along the axon, specifically at the unmyelinated axon segment next to the nerve terminal, are essential for tuning propagated action potentials. Na_v channel clusters near the nerve terminal and their location as a function of neuronal position along the mediolateral axis are controlled by auditory inputs after hearing onset. Thus sound-mediated neuronal activity influences the tonotopic organization of Na_v channels at the nerve terminal in the auditory brain stem.

Abstract

In auditory pathways, the precision of action potential (AP) propagation depends on axon myelination and high densities of voltage-gated Na (Na_v) channels clustered at nodes of Ranvier. Changes in Na_v channel expression at the heminode, the final node before the nerve terminal, can alter AP invasion into the presynaptic terminal. We studied the activity-dependent formation of Na_v channel clusters before and after hearing onset at postnatal day 12 in the rat and mouse auditory brain stem. In rats, the Na_v channel cluster at the heminode formed progressively during the second postnatal week, around hearing onset, whereas the Na_v channel cluster at the nodes was present before hearing onset. Initiation of heminodal Na_v channel clustering was correlated with the expression of scaffolding protein ankyrinG and paranodal protein Caspr. However, in whirler mice with congenital deafness, heminodal Na_v channels did not form clusters and maintained broad expression, but Na_v channel clustering was normal at the nodes. In addition, a clear difference in the distance from the heminodal Na_v channel to the calyx across the mediolateral axis of the medial nucleus of the trapezoid body (MNTB) developed after hearing onset. In the medial MNTB, where neurons respond best to high-frequency sounds, the heminodal Na_v channel cluster was located closer to the terminal than in the lateral MNTB, where neurons respond best to low-frequency sounds. Thus sound-mediated neuronal activities are potentially associated with the refinement of the heminode adjacent to the presynaptic terminal in the auditory brain stem.

NEW & NOTEWORTHY Clustering of voltage-gated sodium (Na_v) channels and their distribution along the axon, specifically at the unmyelinated axon segment next to the nerve terminal, are essential for tuning propagated action potentials. Na_v channel clusters near the nerve terminal and their location as a function of neuronal position along the mediolateral axis are controlled by auditory inputs after hearing onset. Thus sound-mediated neuronal activity influences the tonotopic organization of Na_v channels at the nerve terminal in the auditory brain stem.

in the auditory brain stem circuits, the high density of Na_v channels clustered at discrete subcellular regions is necessary for rapid conduction, which is critical for sound localization circuits that detect timing differences of sounds. Particularly, the high density of Na_v channels at the heminode before the nerve terminal is critical for the reliability and temporal fidelity of presynaptic spikes (Berret et al. 2016; Kim et al. 2013a). The unmyelinated preterminal axon segment consists of a Na_v channel cluster (the heminode) and a Na_v channel-free region (the post-heminode) adjacent to the calyx terminal (Ford et al. 2015). The calyx of Held is a nerve terminal that forms a glutamatergic synapse with the principal neuron of the medial nucleus of the trapezoid body (MNTB) in the auditory brain stem. Previous studies documented the developmental mechanisms responsible for nodal Na_v channel clustering: paranodal axoglial contact, extracellular matrix, and axonal scaffolding proteins ensure Na_v channel clustering at the nodes of Ranvier (Rasband et al. 1999; Susuki et al. 2013; Zhou et al. 1998). However, the mechanisms that refine the formation and localization of the heminodal Na_v channel cluster adjacent to the presynaptic terminal during postnatal development are poorly understood.

Our recent study demonstrated a specific role for compact myelin in dictating ion channel expression and function at the axon heminode (Berret et al. 2016). In the rat auditory brain stem, myelination begins at postnatal days 7 to 8 (P7-8), whereas myelin basic protein (MBP)-dependent compacting of myelin occurs at P12-16 (Berret et al. 2016). Hearing onset is at ∼P12 in rodents. Although axon myelination begins before hearing onset, its maturation and compaction occur at or immediately after hearing onset. Therefore, both sound inputs and myelination may regulate the refinement of channel expression at the heminode along the calyx axon. In particular, sound-mediated neuronal activities affect the refinement of structural and functional properties of ion channels along the axon in the auditory system (Kuba et al. 2006, 2010). Activity-dependent plasticity of the axon initial segment (AIS) refinement occurs in the avian nucleus laminaris (NL) and in rat hippocampal neurons (Grubb and Burrone 2010; Kuba et al. 2006, 2010), and auditory deprivation increases the size of the AIS in the avian NL (Kuba et al. 2010). In addition, the Na_v channel cluster at the AIS is refined into the mature pattern along a tonotopic map, a process driven by both activity-dependent and -independent mechanisms (Kuba et al. 2014). Tonotopy, the orderly representation of the sound frequency to which neurons are most sensitive, is a fundamental organizing principle of auditory brain circuits. In the auditory brain stem, MNTB principal neurons are topographically organized along a mediolateral axis, with the most medially located cells responding to high-frequency sounds and a progressive transition to lower frequency responsiveness in the laterally located cells. The internode length and node diameter of the calyx axon change systematically according to the location of their terminals along the mediolateral axis, suggesting that anatomical parameters of myelinated axons can be optimized for temporal processing of auditory signals in the auditory pathway (Ford et al. 2015).

We studied heminodal Na_v channel clustering at the unmyelinated axon segment adjacent to the calyx terminal and its physiological relevance to presynaptic spiking before and after hearing onset during rat postnatal development (P6-38) and along the mediolateral axis using immunohistochemistry and electrophysiology. Furthermore, we tested the effect of an absence of sound inputs on the refinement of the heminodal Na_v channel cluster in a deaf animal model, congenitally deaf whirler mice (Green et al. 2013; Mburu et al. 2003). We found that heminodal Na_v channel clustering began to form after hearing onset and that the absence of sound input in whirler mice attenuated specifically heminodal Na_v channel clustering. These results suggest that activity-dependent modification of the heminodal Na_v channels occurs at the nerve terminal after hearing onset.

MATERIALS AND METHODS

Animals.

Long-Evans rat and whirler mice (in a C57BL/6 background) pups of either sex at different time points of development (P6-38 for rats and P20-25 for whirler mice) were used in accordance with protocols approved by the University of Texas Health Science Center San Antonio (UTHSCSA) Institutional Animal Care and Use Committee. Whirler mice were provided by Dr. M. A. Bhat's laboratory (UTHSCSA). The wild-type (WT) mice were littermates of the whirler mice. Identification was based on genotyping, with +/+ individuals used as WT mice.

In vivo auditory brain stem response test.

Auditory brain stem response (ABR) recordings were performed as described previously (Kim et al. 2013b). Briefly, mice were anesthetized with 4% isoflurane and maintained with 2% isoflurane during recording (1 l/min O₂ flow rate). ABR recordings were performed in a sound attenuation chamber (Med Associates, Albans, VT). Subdermal needle electrodes (Rochester Electro-Medical, Lutz, FL) were placed on the top of the head, ipsilateral mastoid, and contralateral mastoid as the active, reference, and ground electrodes, respectively. The signal differences in the ABRs between the vertex and the mastoid electrodes were amplified and filtered (100-5,000 Hz). Acoustic stimuli were generated by an auditory evoked potentials workstation [Tucker-Davis Technologies (TDT), Alachua, FL]. Closed-field click stimuli were presented to the left ear. The signals consisted of a series of amplitude-modulated square waves (duration 0.1 ms, repeat rate 16/s) through TDT multi-field magnetic speakers. The sound stimuli were delivered through a 10-cm plastic tube (Tygon; 3.2-mm outer diameter) at a repeat rate of 16/s. Sound intensities ranged from 90 to 20 dB, with 5-dB decrements, and responses to 512 sweeps were averaged.

Slice preparation.

After rapid decapitation of the mice and rats, the brain stem was quickly removed from the skull and immersed in ice-cold low-calcium artificial cerebrospinal fluid (aCSF) containing the following (in mM): 125 NaCl, 2.5 KCl, 3 MgCl₂, 0.1 CaCl₂, 25 glucose, 25 NaHCO₃, 1.25 NaH₂PO₄, 0.4 ascorbic acid, 3 myoinositol, and 2 Na-pyruvate, pH 7.3–7.4 when bubbled with carbogen (95% O₂-5% CO₂) and 310–320 mosmol/l. The brain stem was sectioned (200 μm thick for electrophysiological recordings and 100–120 μm thick for the immunostaining), and the slices were transferred to an incubation chamber containing normal aCSF bubbled with carbogen, where they were maintained for 30 min at 35°C and thereafter at room temperature (24°C). Normal aCSF was the same as low-calcium (slicing) aCSF, but with 1 mM MgCl₂ and 2 mM CaCl₂.

Electrophysiology.

Slices were perfused with normal aCSF at 2 ml/min and visualized using an infrared differential interference contrast microscope (AxoExaminer; Zeiss, Oberkochen, Germany) with a ×63 water-immersion objective and a complementary metal-oxide semiconductor camera (ORCA-Flash2.8; Hamamatsu, Japan). Whole cell patch-clamp recordings were performed in normal aCSF at room temperature (24°C) with the use of an EPC-10 amplifier controlled by PATCHMASTER software (HEKA Elektronik, Lambrecht/Pfalz, Germany). For presynaptic recordings, the pipette solution contained (in mM) 125 K-gluconate, 20 KCl, 5 Na₂-phosphocreatine, 10 HEPES, 4 Mg-ATP, 0.2 EGTA, and 0.3 GTP, pH adjusted to 7.3 with KOH. Recordings were not corrected for the predicted liquid junction potential of 11 mV. Patch electrodes had resistances of 4–5 MΩ. Current-clamp recordings were continued only if the initial uncompensated series resistance was <20 MΩ (Kim et al. 2013a). Alexa568 (50 μM; Invitrogen) was added to the pipette solution to visualize the calyx of Held terminal. Presynaptic APs from the calyx of Held terminal were evoked by stimulation with a bipolar platinum-iridium electrode (Frederick Haer, Bowdoinham, ME) placed near the midline spanning the afferent fiber tract of the MNTB. An Iso-Flex stimulator driven by a Master 10 pulse generator (A.M.P.I., Jerusalem, Israel) delivered 100-μs pulses at 1.2 times threshold (constant voltage <15 V). The average threshold of stimulation intensity was 1.4 ± 0.23 V. Signals were filtered at 2.9 kHz and acquired at a sampling rate of 10–50 μs. Data were analyzed offline and presented using Igor Pro (WaveMetrics, Lake Oswego, OR). Presynaptic AP trains were obtained by averaging three sweeps (5 for a single AP) in each experiment.

Immunohistochemistry.

Rat or mouse brain stem slices (100–120 μm) were fixed with 4% (wt/vol) paraformaldehyde in PBS for 20 min. Free-floating sections were blocked in 4% goat serum and 0.3% Triton X-100 in PBS for 1 h. Slices were incubated with the primary antibody overnight at 4°C. The following primary antibodies were used: mouse anti-sodium channel (PanNa_v; 1:200; Sigma), rabbit anti-calretinin (CR; 1:200; Invitrogen), guinea pig anti-Caspr or rabbit anti-Caspr (Caspr; 1:1,000; obtained from the laboratory of Dr. M. A. Bhat UTHSCSA), mouse anti-ankyrinG (AnkG; 1:100; Neuromab), and guinea pig polyclonal anti-vesicular glutamate transporter 1 (vGluT1; 1:1,000; Millipore). Antibody labeling was visualized by incubation with appropriate Alexa dye-conjugated secondary antibodies (1:500; Invitrogen) for 2 h at room temperature. Stained slices were viewed with laser lines at 488, 568, and 647 nm using a ×63/1.40 NA oil-immersion objective on a confocal laser scanning microscope (LSM-710; Zeiss). Stack images were acquired at a digital size of 1,024 × 1,024 pixels with optical section separation (z interval) of 0.5 μm and were later cropped to the relevant part of the field without changing the resolution. The images were constructed in two dimensions from maximum-intensity projections of confocal image stacks and were imported into MetaMorph for analysis. We drew a box starting from the calyx terminal to the distal axon and measured the fluorescence intensity values within this area. At each pixel (1 pixel = 0.138 μm), the fluorescence intensity values were averaged and then normalized between 0 and 1. The length of the region with Na_v channel expression was determined on the basis of a normalized profile of >0.33 from the starting point to the ending point. The length of the Na_v channel cluster was determined on the basis of a normalized profile of >0.67, whereas the region of broad Na_v channel expression was determined on the basis of a normalized profile between 0.33 and 0.67 (see Fig. 1D). The distance from the Na_v channel at the heminode to the calyx terminal was measured from the point at the outer edge of the principal neuron at the neck of the calyx terminal to the Na_v cluster at the heminode, and the internode length was measured from mid-node to mid-node. The calyx of Held was reconstructed in three dimensions from confocal image stacks using Amira 3D software (FEI, Waltham, MA) and then used to determine the edge as the start of the calyx fingers.

Fig. 1.

The heminodal Na_v channel cluster forms later in development than nodal Na_v clusters in the calyx axon in the MNTB. A: expression of Na_v channels (PanNa_v; green) along the calyx of Held axon (Alexa568 at P8 and P12 or CR at P16; red) in the MNTB of the rat brain stem. White arrows indicate Na_v channel expression, yellow arrows indicate the Na_v channel cluster at the heminode in the calyx of Held, and white dashed circles indicate Na_v channel clusters at nodes. Scale bar, 10 μm. B: Na_v channel expression (PanNa_v; green) at heminodes and nodes during postnatal development (P8-38). The calyx terminal was visualized by Alexa568 at P8 and P12, vGluT1 at P10, and CR staining from P14 to P38. Symbols are as defined in A. Scale bars, 10 μm. C: diagram of the calyx axon, including the heminode containing the Na_v channel cluster and the broad region of expression of Na_v channels that stretches out to the terminal (noncluster). D, top: Na_v channel expression along the Alexa568-filled axon at P12. Yellow dashed line indicates the area in which fluorescence intensity was analyzed. Bottom, normalized fluorescence intensity of PanNa_v in the indicated area in top image. E: bar graph represents the mean length of Na_v channel expression at the node and at the heminode for the Na_v channel cluster and the nonclustering region with broad expression of Na_v channels. ANOVA test: P = 0.022. Error bars indicate SE.

Statistical analysis.

Data values are means ± SE. Immunostaining data were based on analyses from at least six cells in six slices from three to five animals. Experimental data were analyzed and presented using Igor Pro and Prism 5 (GraphPad Software, San Diego, CA). For statistical significance, we tested the normality of the data distribution with the Kolmogorov-Smirnov test with the Dallal-Wilkinson-Lillie correction for corrected P values using Prism 5. If a data set passed the normality test, we used the unpaired Student's t-test; for all other data sets, we used the nonparametric Mann-Whitney U-test. For statistical significance of multiple age groups, we used an ANOVA. For all analyses, P values <0.05 were considered significant.

RESULTS

Na_v channel cluster formation at the calyx of Held axon in the MNTB during postnatal development.

To examine the formation of Na_v channel clusters at the calyx of Held axon in the MNTB during postnatal development, we performed immunostaining for the Na_v channel at the nodes and the heminode using antibodies against PanNa_v, vGluT1, and calretinin (CR; a Ca²⁺-binding protein in the calyx of Held) in P6, P8, P10, P12, P14, P16, P20, and P38 rats. In calyces from young rats (P8 and P12), we performed whole cell patch clamp on the calyx terminal, which was filled with the dye Alexa568 to visualize a single calyx axon, and then performed postrecording immunostaining to identify Na_v channel expression at the heminode and nodes (Fig. 1A). Nodal Na_v clusters were detected in the first postnatal week. The length of nodal Na_v clusters progressively decreased during postnatal development from 1.9 ± 0.14 μm at P8 to 1.1 ± 0.09 μm at P38 (ANOVA test, F = 2.656, P = 0.022; Fig. 1, B and E).

At P6, there was no detectable immunostaining for Na_v channels at the heminode (data not shown). At P8-10, heminodal Na_v channel expression appeared as a sparse band that extended to the calyx terminal (length: 48.1 ± 4.87 μm at P8, n = 6, and 29.4 ± 2.99 μm at P10, n = 20) and within which the normalized fluorescence intensity was between 0.33 and 0.67. At P12-14, Na_v channels began to form clusters at the distal end of the heminode adjacent to the last internode (Fig. 1, B–E). During this period, clustering of heminodal Na_v channels was initiated, and thus both a clustering region with relatively high-intensity staining for Na_v channels (normalized fluorescence intensity > 0.67; length: 3.8 ± 0.36 μm at P12, n = 11, and 2.7 ± 0.21 μm at P14, n = 10) and a longer band of weaker expression were visible in the same axon (length of the weaker band: 25.7 ± 3.55 μm at P12, n = 11, and 16.8 ± 1.86 μm at P14, n = 10; Fig. 1E). After P16, the heminodal Na_v cluster was apparently stabilized (length: 4.6 ± 0.37 μm, n = 9) and kept its compact cluster structure through P38 (3.6 ± 0.26 μm, n = 12), and the broad band of weak staining was no longer detected. Thus the formation of the Na_v channel cluster at the heminode occurred in the second week of postnatal age, considerably later than those that formed at the distal nodes of Ranvier, which appeared during the first postnatal week (ANOVA test, F = 5.801, P = 0.002; Fig. 1, B and E). The data suggest that the initially broad expression of Na_v channels at the heminode coalesces into a cluster during the second week of postnatal age, around the onset of hearing.

Heminodal Na_v channel clustering is correlated with AnkG and Caspr expression.

We next examined the colocalization of heminodal Na_v channel clusters with scaffolding protein at the calyx axon during postnatal development. The axonal cytoskeleton scaffolding protein AnkG is a key molecule in the regulation of Na_v channel clustering and nodal formation in the central nervous system (CNS) (Rasband et al. 1999; Susuki et al. 2013; Zhou et al. 1998). We examined the expression of AnkG at the node and the heminode along the calyx axon at different postnatal ages of rats. At P8-10, when Na_v channels were sparsely expressed at the heminode, AnkG immunoreactivity was not detected (data not shown). At P12-14, when Na_v channels began to cluster at the heminode, AnkG was detected and overlapped with the distal end of the heminodal Na_v channel cluster but was not detected within the region of weak and broad expression of Na_v channels along the heminode (Fig. 2A and data not shown). At the same age, at the nodes of Ranvier, AnkG extended beyond the nodal Na_v cluster into the paranodal regions. Nodal Na_v clusters were detected and corresponded with the middle of AnkG staining (Fig. 2A). Some AnkG expression did not accompany Na_v channel expression along the calyx axon at P12-14. At P20, AnkG was tightly colocalized with the Na_v channel cluster at the heminode and nodes, but its expression also extended through the paranode (Fig. 2B). Na_v clusters were not detected in the absence of AnkG along the calyx axon at this postnatal age. This result suggests that AnkG expression is spatially associated with the formation of Na_v channel clusters at the heminode as well as for stabilizing Na_v channel clusters at the node and heminode during early postnatal development.

Fig. 2.

Expression of AnkG and Caspr at the heminode during development. A: expression of Na_v channels (PanNa_v; green) and AnkG (blue) along the calyx of Held axon (labeled with Alexa568; red) at P12. Dashed yellow lines indicate Na_v channel expression, and arrows indicate Na_v channel clusters; note the regions of overlap with AnkG expression. White dashed circles indicate nodes. Scale bars, 10 μm. Inset: higher magnification image of the node indicated by the circle. Scale bars, 2 μm. B, left: at P20, CR immunostaining (blue) visualizes the calyx axon with Na_v channels (PanNa_v; green) and AnkG (red). The node (circle) and heminode (box) are indicated. Right, higher magnification images of the node and heminode. Arrows indicate Na_v channel clusters and colocalized AnkG. Yellow dashed lines indicate AnkG expression extending to the paranode. C and D, left: Caspr (red) immunostaining with Na_v channels (PanNa_v; green) along the calyx of Held visualized with vGluT1 at P14 (blue) and CR at P20 (blue). Boxes indicate heminodes, and circles indicate nodes. Scale bars, 10 μm. Right, higher magnification images of the node and heminode. Yellow dashed brackets indicate the extent of Caspr expression at the heminode. Yellow arrows indicate the Na_v channel cluster at the node and heminode.

Heminodal Na_v clustering was influenced by the presence of the paranodal protein Caspr as well as AnkG during postnatal development. Caspr is a prominent component of the paranodal axon glial junctions (Bhat et al. 2001). At P8-10, although nodal Na_v channel clusters are well defined by a pair of Caspr-positive paranodes, Caspr was not detectable at the edge of the heminode, within which Na_v channels were expressed throughout a broad region (data not shown). From P12-14, when the heminodal Na_v channel cluster was present adjacent to the last internode, Caspr was expressed at the paranode and formed a border next to the heminodal Na_v channel cluster (Fig. 2C). These findings support the idea that the paranodal axon glial junctions restrict the dispersion of Na_v channels and facilitate Na_v channel clustering at the heminode in the second postnatal week. At P20, Caspr was present at one edge of the Na_v channel cluster at the heminode and was strongly expressed at both edges of nodal Na_v channel clusters (Fig. 2D). Thus the heminodal Na_v clustering process is associated with the formation of the paranodal protein Caspr and the expression of the scaffolding protein AnkG.

Sound stimulation regulates Na_v channel clustering at the heminode after hearing onset.

To test whether sound-evoked neuronal activities affect the refinement of activity-dependent Na_v clustering at the heminode, we used a deaf animal model, the whirler mouse. Whirler (whrn^wi/wi) mice carry a naturally occurring mutant of whirlin (DFNB31), which plays an important role in forming the hair bundle of sensory hair cells in the cochlea, and have profound hearing loss (Lane 1963). Before immunostaining brain stem slices, we confirmed that there were no auditory responses to sound stimulation for whirler mice by using the ABR test. In response to click stimulation, ABRs from WT (whrn^+/+) mice displayed five distinct waves, but whirler mice did not show any detectable waves through the whole range of stimulation (20–90 dB; Fig. 3A), confirming that whirler mice cannot hear. We detected Na_v channel expression at the heminode and nodes along the calyx of Held axon and measured the length of Na_v channel expression in WT and whirler mice (Fig. 3, B and C). In WT mice (at P21), both nodal and heminodal Na_v clusters were apparently stabilized (length: 1.5 ± 0.07 μm, n = 12 for the node vs. 4.4 ± 0.48 μm, n = 10 for the heminode). In whirler mice (at P21), the length of nodal Na_v clusters was 1.4 ± 0.08 μm (n = 16; unpaired t-test, P = 0.08), which was similar to that of WT mice. However, the length of heminodal Na_v clusters in whirler mice was significantly longer than in WT mice (13.7 ± 1.66 μm, n = 20; unpaired t-test, P = 0.0006; Fig. 3D). This result indicates the possibility that Na_v channel clustering at the heminode, rather than at the nodes, is affected by sound stimulation after hearing onset.

Fig. 3.

The heminodal Na_v channel cluster is altered in whirler mice. A: ABR recordings using click stimulation from 90 to 20 dB in WT and whirler mice at P21. B: expression of PanNa_v (green) and CR (red) at the calyx of Held from WT and whirler mice at P21. Symbols are as defined in Fig. 1A. C: higher magnification image of heminodal Na_v channel expression from B and nodal Na_v cluster from WT and whirler mice at P21. Scale bar, 10 μm. D: bar graph represents the mean length of Na_v channel expression at the heminode and node from WT and whirler mice. Unpaired t-test: ***P < 0.001. Error bars indicate SE.

Subcellular location of the heminodal Na_v channel cluster varies along the mediolateral axis in the MNTB.

Expression of ion channels along the mediolateral axis is important for the specialization of neurons with respect to their response to low- or high-frequency sounds (Brew and Forsythe 2005). We next tested whether the size and location of the Na_v channel cluster at the heminode of individual neurons depend on whether the axon terminates in either the medial (responding to high-frequency sound) or lateral (responding to low-frequency sound) region in the MNTB (Fig. 4A). Immunostaining of the MNTB with PanNa_v and CR indicated the length and diameter of the heminodal Na_v channel cluster, as well as the distance from the calyx terminal to the Na_v channel cluster in rats at P16–P25, when Na_v channel clusters are completely stabilized at the heminode (Fig. 4, B and C). The medial calyces were selected from an area located within ∼100 μm of the medial end of the MNTB, whereas the lateral calyces were located within ∼100 μm of the lateral end of the MNTB. The lengths and diameter of Na_v channel clusters at the heminode were similar in the medial and lateral MNTB regions. The mean distance from the calyx terminal to the Na_v channel cluster at the heminode was significantly shorter in the medial MNTB region than in the lateral MNTB region (medial: 10.1 ± 0.61 μm, n = 54 vs. lateral: 20.0 ± 0.96 μm, n = 40; unpaired t-test, P < 0.0001, Fig. 4D). We then examined whether the distance between Na_v channel clusters in the calyx axon was influenced by the mediolateral distribution. The length of the internode between the last node and the heminode was significantly shorter in the lateral area (27.13 ± 2.09 μm, n = 24) than in the medial area (39.8 ± 2.26 μm, n = 27; P < 0.001). In contrast, the lengths of the other internodes (e.g., the second or third internode from the terminal within the MNTB area) in both medial and lateral areas were not significantly different (medial: 43.0 ± 2.32 μm, n = 12 vs. lateral: 49.3 ± 2.46 μm, n = 11; Fig. 4E). The last nodes of the calyx axon before the heminode from either the medial or lateral MNTB region displayed similar lengths (medial, 1.5 ± 0.09 μm, n = 18 vs. lateral, 1.6 ± 0.10 μm, n = 13) and diameters (medial: 1.8 ± 0.16 μm, n = 18 vs. lateral; 1.8 ± 0.08 μm, n = 13; Fig. 4F).

Fig. 4.

Location of the heminodal Na_v channel cluster depends on the tonotopic axis in the MNTB. A: tonotopic organization of the MNTB. LSO, lateral superior olive; VCN, ventral cochlear nucleus. B: expression of PanNa_v (green) and CR (blue) at the calyx of Held in the medial and lateral MNTB at P19-25. Yellow arrows indicate Na_v channel clusters at the heminodes and nodes. White arrows indicate the border of the calyx terminal. Dashed white lines indicate the calyx terminal. Scale bars, 10 μm. C: diagram of the calyx axon showing the calyx terminal and the parameters analyzed (a–g). D–F: analysis of parameters a–g as shown in C between medial and lateral MNTB regions. G: length of the Na_v channel cluster at the heminode and the distance from the heminodal Na_v channel cluster to the calyx terminal in medial and lateral MNTB regions at P10, P12, P14, P16, and P20. Unpaired t-test: **P < 0.01; ***P < 0.001; ****P < 0.0001. Error bars indicate SE; numbers in bars indicate n.

Furthermore, we analyzed the length of Na_v channel clusters at the heminode and the distance between the heminodal Na_v channel cluster and the calyx terminal in the medial and lateral MNTB regions during postnatal development. In both areas, heminodal Na_v channel clustering began around P12, and the length of the Na_v channel clusters became shorter to a similar extent through P20. There was no significant difference in the lengths of Na_v clusters in lateral and medial MNTB regions (at P20: 3.4 ± 0.38 μm for medial, n = 17, and 3.5 ± 0.44 μm for lateral, n = 15; P > 0.79; Fig. 4G). As Na_v clustering proceeded, the location of the heminodal Na_v cluster was also moving away from the calyx terminal at later postnatal ages. Analysis of immunostaining data showed that the distance between the heminode and the calyx terminal was significantly longer in the lateral MNTB area, although both were dependent on the postnatal age (at P20, 12.2 ± 1.31 μm, n = 17 for medial, and 19.4 ± 2.20 μm, n = 15 for lateral; P = 0.007; Fig. 4G). Thus, after hearing onset, the heminodal Na_v cluster became more distant from the calyx terminal in those axons projecting to the lateral region than for the calyx axons projecting to the medial MNTB.

Presynaptic AP firing varies with the location of the calyx terminal along the mediolateral axis.

Neuronal excitability can modulate the location of Na_v channels at the AIS (Grubb and Burrone 2010). Similarly, the tonotopic distribution of heminodal Na_v channels in individual calyx axons may be associated with presynaptic membrane excitability. Interestingly, the cochlear nucleus has tonotopic optimization of postsynaptic membrane excitability, showing that cells with high characteristic frequencies displayed higher firing rates (Oline et al. 2016). We thus examined whether the firing pattern of those axon terminals also follows their spatial location across the mediolateral axis in the MNTB. We studied the AP waveform and presynaptic firing from calyx terminals located in the medial and lateral regions of the MNTB after hearing onset (P12-16 rats). During whole cell recording, the calyces were filled with Alexa568 dye to confirm their location and morphology using postrecording immunostaining with antibodies against PanNa_v and Caspr (Fig. 5A). Presynaptic APs were evoked by afferent fiber stimulation at ∼100 μm from the medial edge of the MNTB. There were no significant differences in the resting membrane potential (medial: −69.6 ± 0.33 mV, n = 9; lateral: −70.2 ± 0.56 mV, n = 11) and the amplitude and half-width of the AP waveforms of calyces from the medial and lateral regions (Fig. 5, B and C). The amplitudes of APs were 113.1 ± 1.64 (n = 9) vs. 116.5 ± 2.10 mV (n = 11) in medial and lateral areas, respectively, and the half-widths of APs were 0.35 ± 0.02 (n = 9) vs. 0.38 ± 0.02 ms (n = 11) in medial and lateral areas, respectively. The amplitude of afterdepolarization (ADP; measured from the resting potential to the peak of the ADP) was significantly larger in the calyces projecting to the lateral region (6.2 ± 1.23 vs. 11.4 ± 1.05 mV, n = 9 and 11 in medial and lateral areas, respectively; P = 0.004; Fig. 5C). The amplitude of afterhyperpolarization (AHP; measured from the resting potential to the peak of the AHP) was larger in the calyces in the medial regions (−5.7 ± 1.03 mV in the medial region vs. −2.8 ± 0.83 mV in the lateral region, n = 9 and 11, respectively; P = 0.04; Fig. 5C). The latency of APs, measured as the time between the stimulus artifact and the peak of the AP, was shorter in the calyces located in the medial region (0.6 ± 0.05 ms, n = 9) than in the lateral region (1.3 ± 0.19 ms, n = 11, unpaired t-test, P = 0.0073; Fig. 5C).

Fig. 5.

Presynaptic AP waveform and high-frequency firing along the mediolateral axis in the MNTB. A, left: calyces located in the medial and lateral MNTB (filled with Alexa568 dye; white arrows) were immunostained with vGluT1 at P16. Right, expression of PanNa_v (green) and Caspr (blue) along individual medial and lateral calyx of Held neurons, which were filled with Alexa568 dye during whole cell recording (at P16). Yellow arrows indicate Na_v channel clusters at the heminode and node. Scale bar, 10 μm. B: representative traces of presynaptic APs from calyces located in the medial and lateral MNTB. C: the amplitude, half-width, and latency of presynaptic APs and the amplitude of ADP and AHP. Unpaired t-test: *P < 0.05; **P < 0.01; ***P < 0.001. D: representative traces of presynaptic AP trains (at 200 Hz) from calyces in the medial and lateral MNTB. Red arrows indicate AP failure in the lateral calyx at 200 Hz. E: AP failure rates at various stimulation frequencies from 50 to 500 Hz (percentage of AP failures in 50 pulses). Nonparametric Mann-Whitney U-test: *P < 0.05; **P < 0.01; ***P < 0.001. Error bars indicate SE.

When the calyx axon was stimulated with trains of 50 pulses, both medial and lateral calyces fired APs with very few failures at 50–100 Hz. At higher frequencies (>200 Hz), however, the reliability of medial calyces was significantly higher with a lower AP failure rate (i.e., there were fewer failures) than lateral calyces (Fig. 5, D and E). At 200 Hz, the medial calyces showed an AP failure rate of 2.9 ± 2.64% (n = 9) during the AP train, whereas the lateral calyces showed a failure rate of 41.8 ± 12.49% (n = 12; nonparametric Mann-Whitney U-test, P = 0.009; Fig. 5E). This result suggests that after hearing onset, the firing pattern of the calyx terminals, as it relates to their placement along the mediolateral axis, may be associated with the activity-dependent refinement of the heminodal location during postnatal development.

DISCUSSION

We have demonstrated that the formation of the heminodal Na_v channel cluster adjacent to the presynaptic terminal occurs around hearing onset in the second week of postnatal age, whereas the nodal Na_v channel cluster is stabilized at P8 before hearing onset. The location of the heminodal Na_v channel cluster depends on the axonal projection either to the medial MNTB (responding to high-frequency sound) or to the lateral MNTB (responding to low-frequency sound). The calyx terminals in the medial MNTB fire APs more reliably at high frequency, and their heminodal Na_v channel clusters are closer to the presynaptic terminal. After hearing onset, sound-evoked neuronal activities may play an important role in the refinement of activity-dependent Na_v channel clustering at the heminode in the auditory brain stem.

Sound stimulation is critical for developmental changes in the heminodal Na_v channels adjacent to the calyx of Held terminal.

Na_v clustering at the node and at the heminode in the MNTB can be differentiated on basis of the level of myelin (Berret et al. 2016). The heminodal Na_v cluster adjacent to the nerve terminal is strongly affected by MBP-mediated compact myelination, much more so than the nodal Na_v channel cluster (Berret et al. 2016). Nodal Na_v channels were stably localized and had formed clusters around P7-8, before the onset of hearing, when oligodendrocytes align along the axon as detected by proteolipid protein (data not shown). Heminodal Na_v channels began clustering at P12, around hearing onset, and this clustering became stabilized by P16, suggesting that hearing inputs strongly influence Na_v channel clustering specifically at the heminode adjacent to the calyx terminal. Congenital deafness in whirler mice attenuated Na_v channel clustering at the heminode of the calyx axon, indicating that sound stimulation is critical for Na_v channel clustering at the heminode rather than at the node. Along with a previous study in the avian NL showing that auditory deprivation increased the size of the AIS (Kuba et al. 2010), our results suggest that there is sound-dependent structural plasticity of the most proximal and distal Na_v clusters rather than of the nodal Na_v clusters along the axons. In early sensory development, auditory deprivation or even moderate hearing loss has a profound effect on the response properties of auditory neurons and the auditory circuit (Harrison and Negandhi 2012; Moore et al. 1989), and unilateral conductive hearing loss alters the structure and function of the calyx of Held–MNTB neuron synapse during postnatal development (Grande et al. 2014). Thus auditory inputs regulate axonal protein expression in the unmyelinated preterminal segment adjacent to the presynaptic terminal in the auditory brain stem.

Relevance of developmental changes in the heminodal Na_v channel to presynaptic AP waveforms at the calyx of Held terminal.

Formation of the Na_v channel cluster at the heminode may be associated with the narrowing of the AP width and high-frequency firing at the presynaptic terminal during this developmental period. From P6 to P14, AP amplitudes of calyx of Held terminals are similar, whereas the AP half-width is reduced by approximately two-thirds, and the maximum rate of rise is almost doubled at P12-14 compared with that at P5-7 (Taschenberger and von Gersdorff 2000). Computer simulation and electrophysiological recordings suggest that the subcellular location of the Na_v channel cluster at the nerve terminal modifies AP waveforms, including their amplitude and half-width (Engel and Jonas 2005; Leão et al. 2005). In these models, when the Na⁺ conductance changed from 0 to 0.25 S/cm² at the calyx, the AP half-width and overshooting increased by ∼20% (Leão et al. 2005). In addition, the presence of specific subtypes of Na_v channel, such as Na_v1.6 and Na_vβ4, at the heminode also contributes to a fast and narrow AP followed by the ADP (Berret et al. 2016; Kim et al. 2010). At P8-10, broad expression of Na_v channels expanding to the calyx terminal could affect the AP overshooting and half-width, and also increase the time difference in the AP latency (jitter) in the immature calyx. Presynaptic recordings and immunostaining data suggest the potential relevance of the heminodal Na_v channel clustering in the maturation of the presynaptic AP waveform during early postnatal development. In addition, AP waveform and membrane excitability are critically affected by K⁺ conductance as well as Na⁺ conductance in the MNTB (Elezgarai et al. 2003; Johnston et al. 2008). An increase in voltage-activated K⁺ channel (K_v) currents and acceleration of K_v kinetics also contribute to the narrowing of presynaptic APs and to reliable firing during early postnatal ages (Dodson et al. 2003; Nakamura and Takahashi 2007). K_v3.1 and Na_v1.6 are colocalized at nodes and heminodes along the calyx of Held axon, although K_v3.1 expression is broader and extends to the calyx terminal compared with the Na_v channel cluster at the heminode (Berret et al. 2016). Na_v channel clustering and its spatial restriction at the heminode may affect K_v3.1 activation, which thus facilitates the narrowing of presynaptic APs.

Scaffolding protein and paranodal structure at the heminode.

The extracellular matrix complex, paranodal structures, and axonal cytoskeletal scaffolding proteins are the main mechanisms for Na_v channel clustering at the nodes of Ranvier in the CNS (Rasband et al. 1999; Susuki et al. 2013). AnkG overlapped only with the Na_v channel cluster at the heminode, rather than with the broad region of Na_v channel expression at P12. Thus initiation of AnkG expression at the border of the broad Na_v channel expression region might trigger and stabilize Na_v channel clusters at the heminode. In addition, the stable structure of the Caspr-positive paranodal junction restricts the mobility of axonal proteins and stabilizes Na_v channel clusters (Bhat et al. 2001; Susuki et al. 2013). Our previous study using a myelin-deficient mutant (Long-Evans Shaker rat) showed that the disruption of the paranodal structure alters specifically the heminodal Na_v clusters rather than nodal Na_v clusters (Berret et al. 2016). Interestingly, although distinct paranodal Caspr expression was located next to the Na_v cluster, it was also detected within the broad region of Na_v channel expression in the heminode in immature calyces at P12-14, whereas this more general expression had completely disappeared at P20. One possible explanation for the presence of Caspr at the heminode might be the transient interaction between the heminode and immature oligodendroglia during early development (Rasband et al. 1999). It could also indicate that the refinement of Caspr expression at the heminode is dependent on the extent of myelination.

Tonotopic arrangement of the heminodal Na_v channel cluster and its physiological relevance.

In the MNTB, the topographic arrangement of K_v channel expression is important for the firing properties of topographically organized MNTB neurons and the tuning of auditory information (Brew and Forsythe 2005; Gazula et al. 2010; Leao et al. 2006; Li et al. 2001; von Hehn et al. 2004). The lateral MNTB neurons, with a lower level of K_v3 channels and a higher level of K_v1 channels, do not reliably generate an AP for every current pulse, in contrast to the medial neurons, which have a much lower failure rate (Leao et al. 2006). Unlike K_v channels in the MNTB neurons, the expression of Na_v channels specifically at the axon terminal in the context of tonotopic organization remains largely unstudied. Our results showed that the diameter and length of the heminode and internode adjacent to the calyx terminals (i.e., <100 μm from the terminal, which included the heminode and next 4 nodes) did not significantly differ between the medial and lateral MNTB regions (Fig. 4). In contrast, at a distance of >500 μm from the heminode, the internode length, internodal axon diameter, and node diameter changed systematically with the distance from the synaptic terminal in the calyx of Held and also were affected by the location of the terminals of the individual neurons within the MNTB (Ford et al. 2015). This result suggested that the spatial differentiation in the anatomical structure of afferent axons affects AP invasion into the calyx terminal. In addition, our results in the spatial differentiation of the heminode and postheminodal segment after hearing onset suggest that sound-evoked neuronal activities may potentially affect this tonotopic distribution of Na_v channels in the nerve terminal.

The cochlear nucleus displays tonotopic optimization of postsynaptic membrane excitability, and thus neurons with high characteristic frequencies have more rapid membrane kinetics, undergo less depression, and are more reliable in spike generation compared with neurons with low characteristic frequencies (Oline et al. 2016). In the present study we found that AP waveform and the firing pattern of calyces depended on their location across the mediolateral axis after hearing onset at P12–P16 (Fig. 5). Presynaptic APs from medial and lateral MNTB regions showed that there were significant differences in the ADP, AHP, and the latency of AP without differences in the amplitude and half-width of presynaptic APs. The ADP and the AHP influence the recovery from inactivation of Na_v channels, which can have a large effect on the ability of neurons to sustain their firing (Johnston et al. 2008). Interestingly, the calyx terminal in the medial MNTB was able to fire APs more reliably in a frequency-dependent manner, relative to the calyx terminal in the lateral MNTB from the rat brain stem. These results suggest the possibility that the development of different neuronal activity along the mediolateral axis after hearing onset may affect AP failure at the terminal during sustained firing associated with the tonotopic distribution of heminodal Na_v channel clusters. In the adult gerbil, however, computer simulation of the calyx spikes has suggested that lateral axons have a shorter half-width and larger amplitude than do medial axons, which could maintain the important temporal information necessary for low-frequency sound source localization (Ford et al. 2015). These contradictory data may result from species or age differences. Both rats and gerbils have the medial-to-lateral, high-to-low characteristic frequency (CF) gradient. However, the gerbil shows a pronounced CF representation below 5 kHz, whereas hearing sensitivity in rats and mice is highest in the mid-to-high-frequency range, about 10–30 kHz (Kopp-Scheinpflug et al. 2008). Thus species difference related to their audiograms can cause these contradictory results. In addition, we should note that in our experiments presynaptic APs were triggered by fiber stimulation at only ∼200 μm from the presynaptic terminal, and thus the involvement of conduction velocity, which depends on axonal diameter and internodal length, along the mediolateral axis was, for the most part, limited and precluded from this analysis. The previous study using computer simulations suggested that the larger diameter and shorter internode length along the axon fiber in the lateral MNTB region produces a faster conduction velocity and a more brief AP than in the medial MNTB in adulthood (Ford et al. 2015).

Understanding the structural properties of the heminode and its functional relevance to presynaptic excitability is essential for determining the relationship between the temporal and spatial coding of calyx spiking at the MNTB during postnatal development. These results suggest the possibility that the refinement of the heminode may be associated with presynaptic membrane excitability and firing properties at the calyx terminal in the MNTB. However, the mechanistic link between presynaptic membrane excitability and the location of the heminodal Na_v channel cluster should be further studied using multiple approaches, such as computer simulation.

GRANTS

This work was supported by National Institute on Deafness and Other Communication Disorders Grant R01 DC03157 (to J. H. Kim).

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the authors.

AUTHOR CONTRIBUTIONS

J.X., E.B., and J.H.K. performed experiments; J.X., E.B., and J.H.K. analyzed data; J.X., E.B., and J.H.K. interpreted results of experiments; J.X. and J.H.K. prepared figures; J.X., E.B., and J.H.K. approved final version of manuscript; E.B. and J.H.K. edited and revised manuscript; J.H.K. drafted manuscript.