Deficiency of neural recognition molecule NB-2 affects the development of glutamatergic auditory pathways from the ventral cochlear nucleus to the superior olivary complex in mouse

Developmental Biology 336 (2009) 192–200

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Developmental Biology j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / d e v e l o p m e n t a l b i o l o g y

Deficiency of neural recognition molecule NB-2 affects the development of glutamatergic auditory pathways from the ventral cochlear nucleus to the superior olivary complex in mouse Manabu Toyoshima a, Kunie Sakurai a, Kuniko Shimazaki b, Yasuo Takeda c, Yasushi Shimoda a, Kazutada Watanabe a,⁎ a b c

Department of Bioengineering, Nagaoka University of Technology, 1603-1, Kamitomiokamachi, Nagaoka, Niigata 940-2188, Japan Department of Physiology, Jichi Medical University, Shimotsuke, Japan Department of Clinical Pharmacy and Pharmacology, Kagoshima University, Graduate School of Medical and Dental Sciences, Kagoshima, Japan

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Article history: Received for publication 17 June 2009 Revised 25 September 2009 Accepted 25 September 2009 Available online 7 October 2009 Keywords: Contactin Auditory brainstem Postnatal development Cell adhesion molecule Glutamatergic neuron Synapse formation Calyx of Held Bushy neuron Apoptosis Auditory brainstem response

a b s t r a c t Neural recognition molecule NB-2/contactin 5 is expressed transiently during the first postnatal week in glutamatergic neurons of the central auditory system. Here, we investigated the effect of NB-2 deficiency on the auditory brainstem in mouse. While almost all principal neurons are wrapped with the calyces of Held in the medial nucleus of the trapezoid body (MNTB) in wild type, 8% of principal neurons in NB-2 knockout (KO) mice lack the calyces of Held at postnatal day (P) 6. At P10 and P15, apoptotic principal neurons were detected in NB-2 KO mice, but not in wild type. Apoptotic cells were also increased in the ventral cochlear nucleus (VCN) of NB-2 KO mice, which contains bushy neurons projecting to the MNTB and the lateral superior olive (LSO). At the age of 1 month, the number of principal neurons in the MNTB and of glutamatergic synapses in the LSO was reduced in NB-2 KO mice. Finally, interpeak latencies for auditory brainstem response waves II–III and III–IV were significantly increased in NB-2 KO mice. Together, these findings suggest that NB-2 deficiency causes a deficit in synapse formation and then induces apoptosis in MNTB and VCN neurons, affecting auditory brainstem function. © 2009 Elsevier Inc. All rights reserved.

Introduction In auditory pathways, the brainstem plays essential roles in the integration of binaural inputs, a process that is critical for sound localization. The lateral superior olive (LSO) is the key nucleus for this integration process. The LSO receives excitatory glutamatergic inputs from spherical bushy neurons of the ipsilateral ventral cochlear nucleus (VCN) as well as inhibitory inputs from the medial nucleus of the trapezoid body (MNTB) (Tolbert et al., 1982; Kuwabara et al., 1991). The MNTB relays aural signals from the globular bushy neurons of the contralateral VCN to the ipsilateral LSO. Interaural time delays and differences in sound intensity are evaluated in the LSO by the balance between excitatory input into the LSO from the ipsilateral VCN and inhibitory input from the MNTB (Irvine, 1986; Sanes, 1990). These projections are elaborately formed to preserve tonotopy, which is the systematic organization of characteristic frequency in the auditory nuclei (Caspary et al., 2008).

⁎ Corresponding author. Fax: +81 258 47 9400. E-mail address: [email protected] (K. Watanabe). 0012-1606/$ – see front matter © 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.ydbio.2009.09.043

During development of the auditory brainstem, gross projections from the cochlear nucleus (CN) to the superior olivary complex (SOC) are completed before birth in gerbils (Kil et al., 1995). At postnatal day (P) 0 in rodents, the terminals from the globular bushy neurons that innervate the cell bodies of principal neurons of the MNTB are thin and filamentous in shape; between P8 and P10, before the onset of hearing, they transform into the young calyx of Held (Mikaelian and Ruben, 1965; Eggermont, 1985). Maturation of the calyx of Held, which is the largest synaptic terminal in the mammalian central nervous system and receives mono-innervation from a globular bushy neuron in the VCN, is completed around P14-P16 (Kandler and Friauf, 1993; Kil et al., 1995). In gerbil, structural refinement of synapses between the MNTB and the LSO is completed in the third postnatal week by reducing the number of boutons per arbor (Sanes and Siverls, 1991). In the LSO, the specific morphological alteration and selective loss of dendritic arbors, depending upon tonotopic position, occur after the onset of hearing (Sanes et al., 1992; Rietzel and Friauf, 1998). To understand the molecular mechanism(s) underlying the refinement and maturation of the auditory brainstem pathways, it is essential to elucidate the role of the molecules that are preferentially expressed in the brainstem after birth.

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NB-2, also referred to as contactin 5, is a neural cell recognition molecule in the contactin subgroup and is preferentially and transiently expressed in developing auditory pathways just before the onset of hearing (Ogawa et al., 2001; Li et al., 2003; Toyoshima et al., 2009). NB-2 knockout (KO) mice exhibit abnormal responses to auditory stimuli in both neuronal excitability and behavior (Li et al., 2003). We recently reported that NB-2 in rat is expressed in bushy neurons of the VCN as well as the ventral acoustic stria (VAS), the glutamatergic presynaptic terminals at the LSO and the calyces of Held in the MNTB at the final stages of auditory brainstem development when synaptic refinement and maturation occur (Toyoshima et al., 2009). To elucidate the role of NB-2 in the development of glutamatergic neurons of the auditory brainstem, we evaluated the effect of NB-2 deficiency on synapse formation and neuronal survival in the brainstem. Materials and methods Animals Mice were maintained in a specific pathogen-free animal facility at Nagaoka University of Technology on a 12:12-h light/dark cycle. All studies described were performed on NB-2-deficient mice and their littermates backcrossed with C57BL/6 for 12 generations. We used mice before 1 month of age because age-related hearing loss at high-frequency sound due to degeneration of outer hair cells becomes evident at around 2–3 months of age in C57BL/6 mice and severe between 6 and 12 months (Willott and Turner, 1999). Protocols and the number of animals used were approved by the Committee for Animal Experiments of Nagaoka University of Technology, and mice were treated in accordance with the Guidelines for the Care and Use of Laboratory Animals of the Japan Society for Neuroscience. For immunohistochemistry and in situ hybridization, mice were anesthetized with pentobarbital and transcardially perfused with phosphate-buffered saline (PBS) for a vascular rinse, followed by 4% formaldehyde in 0.1 M phosphate buffer. Brains were removed and post-fixed in the same fixative overnight at 4 °C and then cryoprotected in 30% sucrose in PBS overnight at 4 °C. For auditory brainstem response (ABR) measurements, mice were anesthetized with isoflurane (4% for induction, 2% for maintenance).

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Immunohistochemistry Mouse brain blocks embedded in O.C.T. compound (Sakura Finetek, Torrance, CA) were cryosectioned at 12 μm, mounted on aminopropylsilane-coated glass slides and air-dried at room temperature. For NB2 immunohistochemistry, the sections were treated with 0.3% H2O2 in PBS, rinsed three times in PBS and then incubated in PBS containing 10% bovine serum albumin and 6% normal horse serum for 1 h at room temperature. Incubation with primary antibodies was performed in PBS containing 1% bovine serum albumin overnight at 4 °C and followed by three rinses in PBS. The sections were incubated with biotinylated antirat IgG in 1.5% normal horse serum for 1 h at room temperature and then rinsed several times in PBS. Vectastain Standard Elite ABC kit (Vector Laboratories, Burlingame, CA) and Tyramide Signal Amplification kit (Invitrogen) were used for immunofluorescence. The sections were incubated with ABC reagent for 30 min at room temperature, rinsed with PBS and finally incubated with Alexa Fluor 488- or Alexa Fluor 546-labeled tyramide in amplification buffer for 5 min at room temperature. After four rinses with PBS, the sections were coverslipped for examination. NB-2 immunostaining with 3,3′-diaminobenzidine has been described previously (Toyoshima et al., 2009). For VGLUT1, Kv1.1, active caspase-3, MAP2 and synapsin 1 immunohistochemistry, sections were incubated with primary antibodies overnight at 4 °C and then with Alexa Fluor 405-, 488- or 546-conjugated secondary antibody for 1 h at room temperature. In situ hybridization A mouse NB-2 cDNA fragment encoding Ig domains III–VI in the pGEM-11Zf(+) vector (Promega, Madison, WI) was used to prepare RNA probes, which were labeled with digoxigenin (DIG) using the DIG RNA Labeling kit (Roche, Basel, Switzerland). The procedures and conditions for in situ hybridization have been described (Toyoshima et al., 2009). Image analysis Images were acquired on an Axiovert 200 microscope (Carl Zeiss Microimaging, Inc., Thornwood, NY) or a Fluoview FV1000 confocal

Antibodies Monoclonal antibodies 1B10 and 1C4 against mouse NB-2 were generated by the rat lymph node method (Kishiro et al., 1995) in Wistar rats immunized with the extracellular domain (amino acids 1– 1065) of the mouse NB-2 protein fused to the Fc portion of human IgG (NB-2-Fc). 1B10 was used for western blot analysis and immunoprecipitation. 1C4 was used for immunohistochemistry. The other primary antibodies used were anti-vesicular glutamate transporter 1 (VGLUT1) (1:2,500, guinea pig polyclonal; Chemicon, Temecula, CA), anti-Kv1.1 (1:50, mouse monoclonal; NeuroMab, Davis, CA), antiactive caspase-3 (1:1,000, rabbit polyclonal; BD Biosciences, San Jose, CA), anti-microtubule associated protein-2 (MAP2) (1:1,000, mouse monoclonal; Chemicon), anti-MAP2 (1:1,000, rabbit polyclonal; Chemicon) and anti-synapsin 1 (1:1,000, mouse monoclonal; Synaptic Systems, Goettingen, Germany). Secondary antibodies were Alexa Fluor 488-conjugated anti-mouse IgG (1:2,000; Invitrogen, Carlsbad, CA), Alexa Fluor 546-conjugated anti-mouse IgG (1:2,000; Invitrogen), Alexa Fluor 405-conjugated anti-rabbit IgG (1:2,000; Invitrogen), Alexa Fluor 488-conjugated anti-rabbit IgG (1:2,000; Invitrogen), Alexa Fluor 546-conjugated anti-rabbit IgG (1:2,000; Invitrogen) and Alexa Fluor 546-conjugated anti-guinea pig IgG (1:2,000; Invitrogen).

Fig. 1. Characterization of rat anti-mouse-NB-2 monoclonal antibodies 1C4 and 1B10. (A) Sagittal brain sections from NB-2 KO and wild-type (WT) mice at P5 were immunostained with 1C4. Strong immunoreactivity was detected in the anteroventral thalamus (AV), olfactory bulb (OB), corpus callosum (cc) and the posterior half of the cerebral cortex along with the superior olivary complex (SOC) and inferior colliculus (IC) in the wild-type mouse. This staining profile was similar to that of rat brain using mouse anti-rat-NB-2 monoclonal antibody 3G12. No staining was observed in the KO mouse. (B) Reactivity of the 1C4 and 1B10 antibodies was examined in western blots of brain lysates from NB-2 KO and wild-type mice. 1B10 produced a single band at approximately 130 kDa (corresponding to the size of NB-2) in brain lysate from the wild-type mouse, but not from the NB-2 KO mouse. 1C4 gave a weak band in brain lysate from the wild-type mouse, but not from the NB-2 KO mouse. Scale bar: 1.5 mm.

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Fig. 2. NB-2 expression in the VCN and SOC in mouse at P6. A schematic drawing of the auditory pathway from the VCN to SOC in mouse (A). Auditory signals generated in the cochlea are carried to the cochlear nucleus. Signals from the ventral cochlear nucleus (VCN) travel to the superior olivary complex (SOC), which comprises four nuclei (LSO, SPN, MSO, MNTB). The lateral superior olive (LSO) receives excitatory inputs from ipsilateral VCN as well as inhibitory inputs from the medial nucleus of the trapezoid body (MNTB). The MNTB relays aural signals from the contralateral VCN to the ipsilateral LSO. The LSO integrates binaural inputs and sends signals to the inferior colliculus (IC). Strong NB-2 immunoreactivity (green) was observed in the VCN (B, C), VAS (C) and nuclei of the SOC (D) at P6. VGLUT1 signals (magenta) overlapped with NB-2 signals in the LSO (arrowheads in E) and calyces of Held in the MNTB (arrows in F). In situ hybridization for NB-2 mRNA with antisense probe (G) revealed positive neurons in the PVCN, whereas the corresponding sense probe (H) did not. NB-2 in situ hybridization signals (green) co-localized with Kv1.1 immunohistochemistry signals (magenta; I, J), indicating that NB-2 is expressed in bushy neurons of the VCN. AVCN, anteroventral cochlear nucleus; LSO, lateral superior olive; MNTB, medial nucleus of the trapezoid body; PVCN, posteroventral cochlear nucleus; SPN, superior paraolivary nucleus; VAS, ventral acoustic stria. Scale bars: 200 μm in B–D, G–I; 20 μm in E, F, J.

laser scanning microscope (Olympus, Tokyo, Japan). Confocal images were converted to TIFF format with FV10-ASW software (Olympus). Light microscopic images were acquired with a DP70 digital camera and Controller and Manager software (Olympus). Images were compiled manually in Adobe Photoshop CS2 (Adobe Systems, San Jose, CA). Further image processing (to optimize brightness, contrast and color balance) was performed in Adobe Photoshop CS2, if necessary. For morphometric analysis, the outlines of immunopositive area were traced digitally, and their areas were determined using MetaMorph software (Molecular Devices, Downingtown, PA).

Auditory brainstem responses Calibrated click stimuli at 8, 12, 16 and 20 kHz were applied to anesthetized mice. Subcutaneous steel-tipped electrodes were placed at bilateral vertices, and a ground electrode was attached to the back of the mouse. Click stimuli were generated by an Rp2.1 system (TuckerDavis Technologies, Alachua, FL) and delivered to the left ear in 5-dB steps from 10 to 90 dB by placing a speaker inside the pinna. The rise/ fall times for the tone bursts were 0.1 ms rise/ms flat. The electroencephalogram in response to click stimuli was obtained as

Fig. 3. Reduction of principal neurons with VGLUT1-positive calyces in the MNTB of NB-2 KO mouse. (A) The calyces of Held were co-immunostained with markers for principal neurons (MAP2, blue), presynaptic terminals (synapsin 1, green) and mature glutamatergic synapses (VGLUT1, red). MAP2 signals that lacked VGLUT1 signals, but harbored synapsin 1 signals, were detected in wild-type and NB-2 KO mice at P6 (arrows). MAP2 signals that lacked both synapsin 1 and VGLUT1 were observed only in NB-2 KO at P6 (arrowheads). Almost all MAP2 signals were encircled with both synapsin 1 and VGLUT1 signals in NB-2 KO and wild-type mice at M1. (B) A substantial number of MAP2-positive cells without synapsin 1 signals were detected in NB-2 KO, but not in wild type, at P6. The number of such cells was negligible in both genotypes at M1. (C) The ratio of synapsin 1 signals without VGLUT1 signals was significantly increased at P6, but not at M1, in NB-2 KO mice as compared to wild type. (D) The number of MAP2-positive cells was compared between NB-2 KO and wild-type mice. We counted the number of MAP2-positive cells in images of 50 brainstem sections from five NB-2 KO and five wild-type mice and then calculated the average number of cells per section. At M1, the number of MAP2-positive cells was 10% lower in NB-2 KO mice compared to wild type. (E) The size of the MNTB was compared between NB-2 KO and wild-type mice. The average area at M1 was 14% smaller in NB-2 KO mice than wild-type mice, whereas no significant difference was detected at P6. (F) The size of principal neurons was compared between NB-2 KO and wild-type mice. Significant difference was not detected in area of principal neurons between two genotypes at both P6 and M1. Scale bars: 50 μm. ⁎p b 0.05, ⁎⁎p b 0.01 and ⁎⁎⁎p b 0.001 (Student's t-test).

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differentially recorded scalp potentials using a digital averaging system (PowerLab system, AD Instruments, NSW, Australia). At each sound level, 500 responses to click stimuli were averaged to obtain the ABR signal. ABR waves I–V were assessed as the click stimulus was increased from 10 to 90 dB. The lowest stimulus level that yielded a detectable ABR waveform was defined as the threshold. Interpeak latencies (IPLs) were defined as the time intervals between ABR waves.

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Results Generation and characterization of rat anti-mouse NB-2 monoclonal antibodies Because the mouse anti-rat-NB-2 monoclonal antibody 3G12 we previously generated does not react with mouse tissues (Toyoshima

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et al., 2009), we generated anti-mouse-NB-2 monoclonal antibodies 1C4 and 1B10 in rat in a similar manner to 3G12, using a recombinant mouse NB-2-Fc fusion protein for immunization and a recombinant mouse NB-2 tagged with 6× His as an antigen. Antibodies were screened by enzyme-linked immunosorbent assay and western blotting. Immunohistochemical staining of brain sections from wild-type mice at P5 with 1C4 produced strong signals in the SOC and inferior colliculus (IC) of the auditory pathway and in the occipital lobe of the cerebral cortex, including the auditory cortex, anteroventral thalamus and corpus callosum (Fig. 1). The staining pattern of 1C4 in mouse brain was similar to that of 3G12 in rat brain. In contrast, no signal was detected in the brain section from an NB-2 KO mouse. Antibody 1B10 produced no signal in brain sections from an NB-2 KO mouse or wildtype mouse (data not shown). Thus, for immunohistochemistry, 1C4, but not 1B10, specifically reacted with NB-2 proteins in the mouse brain. In a western blot of mouse brain lysates, 1C4 yielded a weak band at approximately 130 kDa, which corresponds to the molecular mass of NB-2 (Fig. 1B). 1B10 gave a strong and clear band at 130 kDa in the brain lysate from wild-type mouse but not from NB-2 KO mouse. These results indicated that 1B10 is suitable for detection of NB-2 in western blots. NB-2 localization in the VCN and SOC of P6 mice Using antibody 1C4, we examined NB-2 immunoreactivity in the mouse VCN and SOC. Fig. 2A shows a schematic drawing of the auditory pathway from the VCN to SOC. During development of the VCN and SOC, NB-2 was expressed transiently, between P1 and P7, with a maximum level at P5 (data not shown). The NB-2 expression profile in mouse was very similar to that in rat (Toyoshima et al., 2009). Briefly, NB-2 was strongly expressed in the VCN and SOC together with the VAS, which is the bundle of fibers projecting from the VCN to the contralateral and ipsilateral SOC (Figs. 2B–D). In the VCN, NB-2 signals decreased gradually along the dorsoventral axis, suggesting that NB-2 is preferentially localized in the tonotopic highfrequency regions of the brainstem in mouse, as discussed in rat (Toyoshima et al., 2009). In the SOC, NB-2 signals co-localized, at least in part, with VGLUT1 signals at the neuropil in the LSO (Fig. 2E) and at the calyces of Held, the large glutamatergic presynaptic terminals from contralateral VCN globular bushy neurons that encircle the somata in the MNTB (Fig. 2F). To determine NB-2-expressing neurons in the VCN, we performed in situ hybridization with NB-2 probes, followed by immunostaining for the potassium channel Kv1.1 as a marker for bushy neurons (spherical and globular) (Pal et al., 2005; Perney and Kaczmarek, 1997). A large proportion of NB-2-positive cells were co-labeled for Kv1.1 (Figs. 2I, J), indicating that NB-2 is expressed in bushy neurons of the VCN. Reduction in calyces of Held and principal neurons in the MNTB of NB-2 KO mice NB-2 was localized in the glutamatergic pathway, from the bushy neurons in the VCN to the ipsilateral LSO and contralateral MNTB in the SOC via the VAS. To evaluate the possible role of NB-2 at developing synapses in this pathway, we examined the development of the calyces of Held in the MNTB. The calyces of Held and the principal neurons in the MNTB are large, and most principal neurons receive mono-innervation from globular bushy neurons of the VCN at the calyx of Held (Hoffpauir et al., 2006). Thus, it was appropriate to examine whether NB-2 deficiency in the globular bushy neurons affected the calyces of Held and the principal neurons in the MNTB. We first compared synapsin 1 and VGLUT1 immunoreactivity of the calyces of Held between NB-2 KO and wild-type mice at P6 and at 1 month (M1) (Fig. 3A). In the wild-type mouse at P6, synapsin 1 and

Fig. 4. Reduced number of VGLUT1-positive signals in the LSO of the NB-2 KO mouse. (A) Sections from the LSO of NB-2 KO and wild-type (WT) mice at P6 were immunostained for VGLUT1. VGLUT1-positive dots in the LSO were counted in 75 sections from five NB-2 KO mice and five wild-type mice. The images were converted to a binary format to count the number of VGLUT1-positive signals. (B) The average number of VGLUT1-positive signals per 400 μm2 was compared between NB-2 KO and wild-type mice at P6 and M1. At P6 and M1, the number of signals was 15% lower in the NB-2 KO mouse relative to wild type. ⁎p b 0.05 (Student's t-test).

VGLUT1 signals in the MNTB mostly overlapped and were restricted to the calyces of Held, displaying a thick labeling pattern around most of the somata of MNTB principal neurons, which were identified by immunostaining with MAP2. A subset of principal neurons was encircled with VGLUT1-negative calyces, which were labeled with synapsin 1 alone (arrows in Fig. 3A). In the NB-2 KO mouse at P6, there was scattering of principal neurons lacking both synapsin 1 and VGLUT1 signals (arrowheads in Fig. 3A), indicating the existence of principal neurons without calyces of Held. Quantitatively, 8.3% ± 1.3% of principal neurons in the MNTB were not encircled by calyces of Held in the NB-2 KO mouse compared to 0.16% ± 0.09% in wild type (p b 0.001, n = 50; Fig. 3B). Furthermore, the ratio of principal neurons with VGLUT1-negative calyces significantly increased in the NB-2 KO mouse (7.7% ± 1.0%) relative to wild type (5.0% ± 0.67%) at P6 (p = 0.023, n = 50; Fig. 3C). In contrast, at M1, the negligible number of principal neurons without calyces of Held was observed in both NB2 KO mice and wild-type mice (p = 0.809, n = 50; Fig. 3B). The ratios of principal neurons with VGLUT1-negative calyces at M1 were much lower than those at P6 in both NB-2 KO mice (1.3% ± 0.59%) and wildtype mice (0.88% ± 0.38%), exhibiting no significant difference between two genotypes at M1 (p = 0.406, n = 50; Fig. 3C). Thus, almost all calyces were positive for VGLUT1 in both NB-2 KO mice and wild-type mice at M1. Next, we compared the number of principal neurons in the MNTB between NB-2 KO and wild-type mice by immunostaining with MAP2. We selected images from five NB-2 KO and five wild-type mice showing the 50 largest cross-sections of the MNTB and counted the number of MAP2-positive cells in each section. From these counts, we obtained the average number of principal neurons in each section. Wild-type and NB-2 KO mice at P6 had an average of 59.4 ± 3.3 and 57.2 ± 2.5 principal neurons per MNTB section, respectively (Fig. 3D). There was no significant difference in the number of principal neurons

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between NB-2 KO and wild-type mice at P6 (p = 0.482, n = 50). At M1, the wild-type mouse had 92.6 ± 3.2 principal neurons per MNTB section, whereas the NB-2KO mouse had 82.4 ± 3.6 principal neurons per section (Fig. 3D). Thus, at M1, the number of principal neurons was 10% lower in the NB-2 KO mouse than in wild type (p = 0.008, n = 50), implying that the number of calyces of Held was also reduced in the NB-2 KO mouse at M1. In addition, we compared the size of the MNTB between NB-2 KO and wild-type mice by determining area containing principal neurons stained for MAP2. The difference between NB-2 KO (31,300 ± 800 μm2) and wild-type mice (32,200 ± 800 μm2) was not significant at P6 (p = 0.488, n = 50; Fig. 3E). At M1, the area of MNTB in NB-2 KO mice (34,000 ± 1,500 μm2) was decreased compared to wild-type mice (39,600 ± 1,400 μm2; Fig. 3E). This significant decrease by 14% in the area of MNTB (p = 0.027, n = 50) corresponded to 10% decrease in the number of principal neurons at M1. On the other hand, there was no significant difference in the cell size of principal neurons between genotypes at both P6 and M1 (p = 0.503 and p = 0.606, respectively, n = 20 cells per mouse from five animals of each genotype; Fig. 3F). Together, these results

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show that NB-2 contributes to synaptic formation between globular bushy neurons in the VCN and principal neurons in the MNTB. Reduction in number of glutamatergic synapses at the LSO in NB-2 KO mice The LSO, which receives excitatory inputs from spherical bushy neurons in the ipsilateral VCN and inhibitory inputs from principal neurons in the MNTB, is another important SOC nucleus that is immunopositive for NB-2. We compared the number of VGLUT1immunoreactive signals at the LSO in 75 images of sections from five NB-2 KO and five wild-type mice (Fig. 4). VGLUT1 immunoreactivity at the LSO reflects excitatory presynaptic terminals of spherical bushy neurons of the VCN. At P6, the number of VGLUT1 signals in 400 μm2 was 107.5 ± 6.3 in the NB-2 KO mouse and 126.3 ± 4.3 in wild type. At M1, the number was 80.2 ± 3.7 in NB-2 KO mouse and 95.3 ± 3.0 in wild type. Thus, the number of VGLUT1 signals in the NB-2 KO mouse was reduced by 15% at P6 and M1 relative to wild type (p = 0.041 and p = 0.022, respectively, n = 75). Thus, NB-2 contributes not only to

Fig. 5. Apoptotic activity in principal neurons in the MNTB and bushy neurons in the VCN of the NB-2 KO mouse. (A) To detect apoptotic activity in the MNTB, cells were immunostained for caspase-3 (magenta) and MAP2 (green) at P10 and P15. We selected 50 section images from five NB-2 KO and five wild-type mice. In both NB-2 KO and wild-type mice, small caspase-3-positive cells were scattered in the MNTB (arrows). In contrast, large caspase-3-positive cells, which were apoptotic principal neurons, were almost exclusively detected in NB-2 KO mice at P15 (arrowheads). (B) The number of small apoptotic cells in the MNTB of NB-2 KO mouse (white bar) did not differ significantly from that of wild type (black bar) at both P10 and P15. (C) The percent of large apoptotic cells in the MNTB was unequivocally higher in the NB-2 KO mouse (white bar) relative to wild type (black bar) at both P10 and P15. (D) Sections from the MNTB of NB-2 KO mice at P15 were triply immunostained for caspase-3 (magenta), VGLUT1 (green) and MAP2 (blue). Large apoptotic cells (arrowheads) were observed independently from VGLUT1-encircled MAP2 signals. (E) Caspase-3-positive cells in the VCN of NB-2 KO and wild-type mice at P15. (F) The number of caspase-3-positive cells in the VCN was significantly higher in the NB-2 KO mouse (white bar) compared with wild type (black bar). Scale bars: 50 μm in A, D, E. ⁎⁎p b 0.01 and ⁎⁎⁎p b 0.001 (Student's t-test).

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the projection from globular bushy neurons to the calyces of Held in the MNTB but also to that from the spherical bushy neurons in the VCN to the LSO. Apoptosis of principal neurons in the MNTB and bushy neurons in the VCN in NB-2 KO mice To investigate the cause for the reduced number of principal neurons in the MNTB of NB-2 KO mice, we examined apoptotic activity in the MNTB (Fig. 5). Immunostaining with caspase-3 detected two types of apoptotic cells. Apoptotic cells that were much smaller than principal neurons were sparsely distributed in the MNTB of both NB-2 KO and wild-type mice (arrows in Fig. 5A). The NB-2 KO mouse did not differ significantly from wild type in the number of small apoptotic cells at either P10 or P15 (Fig. 5B). Few, if any, large apoptotic cells were observed in wild type at P10 or P15. These results are consistent with previous findings that the principal neurons do not undergo apoptosis during normal postnatal development, despite the presence of many small apoptotic non-neuronal cells in the MNTB (Rodriguez-Contreras et al., 2006). Interestingly, many large apoptotic cells were detected only in the NB-2 KO mouse (arrowheads in Fig. 5A). There was a three-fold increase in the ratio of large apoptotic cells to the principal neurons in the NB-2 KO mice from P10 (1.6% ± 0.5%) to P15 (5.9% ± 0.6%), whereas the number of large apoptotic cells remains negligible in wild-type mice at both P10 and P15 (p = 0.009 and p b 0.001, respectively, n = 30). The size of these cells (Rodriguez-Contreras et al., 2006) and their appearance only in the NB-2 KO mouse, which is shown to have fewer principal neurons at M1 (Fig. 3D), suggested that these represent apoptotic principal neurons. In addition, triple immunostaining with caspase-3, MAP2 and VGLUT1 showed that no apoptotic activity (caspase-3 immunoreactivity) was detected in the principal neurons with mature calyces of Held, which were revealed as VGLUT1-encircled MAP2 signals, in the MNTB of NB-2 KO mice (Fig. 5D). Large apoptotic cells were observed (arrowheads in Fig. 5D) as distinct from the principal neurons with mature calyces. Together, these results suggested that the principal neurons lacking mature innervations might undergo apoptosis. We also examined the apoptotic cells in the VCN because a defective calyx from a bushy neuron of the NB-2-deficient VCN could fail to mature and affect the survival of not only the principal neuron but also the bushy neuron. The average number of apoptotic neurons per section of the VCN at P10 was 8.7 ± 0.9 in the NB-2 KO mouse and 4.4 ± 0.7 in wild type (Figs. 5E, F). At P15, the average number was 8.4 ± 0.7 in the NB-2 KO mouse and 4.7 ± 0.5 in wild type (Fig. 5F). Thus, the number of apoptotic cells in the VCN was higher in the NB-2 KO mouse relative to wild type at P10 and P15 (p = 0.003 and p = 0.005, respectively, n = 30). Increase in interpeak latency of ABR waves II–V in NB-2 KO mice To estimate the effects of NB-2 deficiency on auditory brainstem function, we recorded auditory brainstem responses (ABRs) of NB-2 KO and wild-type mice at M1. ABR thresholds at 8, 12, 16 and 20 kHz of NB-2 KO mice, which were at 20–30 dB SPL (sound pressure level) in this frequency range, exhibited little difference from those of wild type (p = 0.292, p = 0.565, p = 0.440 and p = 0.884, respectively, n = 10; Fig. 6A). Fig. 6B shows typical ABR waveforms at 8 kHz for comparison between NB-2 KO and wild-type mice as examples. There was no significant difference in the ABR peak amplitudes of waves I to V between genotypes (p = 0.288, p = 0.688, p = 0.633, p = 0.460 and p = 0.272, respectively, n = 10). On the other hand, the delay of waves III, IV and V in NB-2 KO mice relative to those of wild type was consistently observed. We analyzed the time intervals between waves as interpeak latencies (IPLs). IPLs for waves II–III and III–IV were

Fig. 6. Increase in interpeak latencies between II–III and III–IV in ABR measurements of NB-2 KO mice. ABR threshold and wave latency were examined in NB-2 KO and wildtype mice. (A) The ABR thresholds in response to tone bursts (8, 12, 16 and 20 kHz) were measured in NB-2 KO mice and wild-type mice (WT) at P30. There was no significant difference in threshold between NB-2 KO and wild-type mice. (B, C) By contrast, interpeak latencies between waves II–III and III–IV were significantly increased in NB-2 KO mice. Panel B shows an example of an ABR waveform at 8 kHz from wild-type and NB-2 KO mice. The ABR waves were generated by click stimulus at 80 dB. ⁎p b 0.05 and ⁎⁎p b 0.01 (Mann–Whitney test).

significantly augmented in NB-2 KO mice as compared to wild type (p = 0.016 and p = 0.008, respectively, n = 10; Fig. 6C). Discussion In the present study, we compared the postnatal development of the auditory brainstem between NB-2 KO and wild-type mice to elucidate the contribution of NB-2 in this process. As a prerequisite for this analysis, we examined localization of NB-2 in the auditory brainstem in mouse using the newly generated monoclonal antibody, 1C4 (Fig. 1). We confirmed that localization of NB-2 in the mouse

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auditory brainstem was very similar to its previously reported localization in rat (Toyoshima et al., 2009). NB-2 immunoreactivity was detected in the VCN and VAS as well as the nuclei of the SOC (Figs. 2B–D). The VAS is the bundle of axons projecting from the bushy neurons in the VCN to the ipsilateral LSO and contralateral MNTB. The LSO receives glutamatergic input from ipsilateral spherical bushy neurons of the VCN and the principal neurons of the MNTB receive large glutamatergic synapses (the calyces of Held) from contralateral globular bushy neurons (Tolbert et al., 1982; Kuwabara et al., 1991). In the SOC, NB-2 immunoreactivity was observed at glutamatergic synapses in the LSO and MNTB (Figs. 2E, F). In addition, in situ hybridization demonstrated that NB-2 is expressed in the bushy neurons of the VCN (Figs. 2I, J). Taken together, these results indicate that NB-2 at glutamatergic synapses in the LSO and MNTB is transported from bushy neurons in the VCN via the VAS. NB-2 was expressed transiently in the VCN and SOC between P1 and P7 with a maximum level at P5, before the onset of hearing in mouse (data not shown). Young calyces of Held start to form just after birth and are completed between P8 and P10 in rodents (Mikaelian and Ruben, 1965; Eggermont, 1985). As shown in Fig. 2F, NB-2 partially overlapped with VGLUT1, a marker for mature glutamatergic synapses (Blaesse et al., 2005), in the MNTB at P6. As we have previously shown in rat (Toyoshima et al., 2009), NB-2 expression is followed by VGLUT1 expression in the SOC and declines thereafter. We speculate that NB-2 signals might be no longer detectable at the mature region of calyces where VGLUT1 signals are strong. Therefore, a possible role of NB-2 in the MNTB might be in the initial stage of calyx maturation. Here, we demonstrate that NB-2 deficiency in the mouse causes significant alternation and induces apoptosis in a subset of neurons in the auditory brainstem. In the MNTB at P6, while almost all principal neurons were encircled with calyces of Held in wild-type mouse, principal neurons without calyces were scattered in the NB-2 KO mouse (Fig. 3B), but there was little difference in the number of principal neurons between the two genotypes (Fig. 3D). These results indicate that a subset of principal neurons may not receive the inputs at all in NB-2 KO mice at P6. In addition, the ratio of VGLUT1-negative calyces increased approximately1.5-fold in the NB2 KO mouse relative to wild type (Fig. 3C). Because VGLUT1 is a neuronal marker for mature synapses (Blaesse et al., 2005), this result implies that more principal neurons are encircled by immature calyces in the NB-2 KO than in wild type at P6. Furthermore, the total number of principal neurons was reduced in the NB-2 KO mouse at M1 relative to wild type, although almost all principal neurons were wrapped with VGLUT1-positive calyces of Held in both NB-2 KO and wild-type mice at M1 (Figs. 3B, C). These results suggest that principal neurons without mature innervation from the globular bushy neurons of the VCN are eliminated between P6 and M1. To explore this possibility, we examined apoptotic activity in the MNTB and VCN at P10 and P15. In wild-type mice, small apoptotic cells in the MNTB were detected in P10 and P15, but no large apoptotic cells were observed (Figs. 5A–C). In contrast, apoptotic activity in the principal neurons was detected in the NB-2 KO mouse at P15 and to a lesser extent at P10 (Figs. 5A, C). The apoptotic activity was exclusively observed in the cells without mature innervations (Fig. 5D). Therefore, we suppose that substantial apoptosis in the principal neurons of the NB-2 KO mouse at P15 might result from failure in the formation and/ or maturation of the calyces of Held rather than another direct effect of NB-2 deficiency. The apoptosis observed at P15 would account for the reduced number of principal neurons in the MNTB of the NB-2 KO mouse at M1. In this regard, apoptotic activity was also observed in the VCN in the NB-2 KO mouse at both P10 and P15 (Figs. 5E, F). Most of the individual globular bushy neurons in the VCN innervate a single principal neuron in the MNTB via the calyx of Held. This indicates that a

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defect in either the globular neurons or the principal neurons directly affects survival of the partner neurons. If this role for NB-2 expression in the VCN is consistent, glutamatergic synapses at the LSO should also be affected by apoptotic activity in the VCN. A significant decrease in the number of VGLUT1 signals in the LSO, which reflect excitatory presynaptic termini of spherical bushy neurons in the VCN, was observed in the NB-2 KO mouse not only at P6 but also at M1 (Fig. 4). This finding is consistent with the increase in apoptotic cells observed in the VCN at P10 and P15. Together, these findings strongly suggest that lack of NB-2 expression in globular and spherical bushy neurons in the VCN leads to failure in glutamatergic synapse formation and/or maturation in the LSO and the MNTB and then induces apoptosis of both the presynaptic bushy neurons in the VCN as well as the postsynaptic principal neurons in the MNTB. Although nothing is known on the NB2 signaling mechanism leading to the changes in synapse formation, it should be noted that some contactin subgroup members bind to amyloid precursor protein (APP) family proteins (Osterfield et al., 2008; Ma et al., 2008). Because NB-2 binds to APLP1 in the family (Osterfield et al., 2008) and synapse formation and function is modulated by APP (Ashley et al., 2005; Priller et al., 2006; Hoe et al., 2009), it might be possible to speculate that NB-2 plays a role in synapse maturation coupled with APP signaling. We previously reported that neuronal excitability in the IC monitored by c-Fos expression differs between NB-2 KO and wildtype mice (Li et al., 2003), indicating that function of the pathway from the SOC to the IC is impaired by NB-2 deficiency. Here, we show that the NB-2 KO mouse exhibits a significant increase in the IPLs of ABR waves II–III and III–IV relative to wild type, without detectable difference in the ABR thresholds or peak amplitudes. ABR waves II–III are responses derived from spherical and globular bushy neurons of the VCN and their targets in cat (Fullerton and Kiang, 1990; Melcher et al., 1996; Melcher and Kiang, 1996). Waves III–V are combined responses from the CN, SOC, lateral lemniscus and IC. The latency between waves can be roughly considered an integrated nerve conduction velocity between nuclei. Increased ABR peak latencies have been observed in patients with multiple sclerosis (Keith et al., 1987), suggesting that a defect in myelination may account for the increase in IPLs. However, it is unlikely that NB-2 deficiency affects myelination in the auditory brainstem because NB-2 was transiently expressed between P1 and P7 in the auditory brainstem including the VAS and hardly detectable at the time of myelination (data not shown). In addition, we did not observe any abnormal expression of myelin-associated glycoprotein (MAG), myelin marker, in the VAS of NB-2 KO mouse at P21 (data not shown). These suggest that the increase in the IPLs of NB-2 KO mouse will be influenced by other defects than myelination. In this regard, a decrease in ABR wave III latency occurs when there is a reduction in the magnitude of inhibitory input from MNTB cells to LSO neurons (Saul et al., 2008) or when wave III consists of two components and the amplitude of the slower component is diminished, as shown by brainstem lesion experiments in the cat (Fullerton and Kiang, 1990). Then, we propose that apoptosis of subsets of bushy neurons in the VCN and principal neurons in the MNTB in the NB-2 KO mouse could affect the magnitude of excitatory and inhibitory inputs to LSO neurons from the VCN and MNTB, respectively, and thereby increase the IPLs for waves II–III and III–IV. By the failure in synapse formation at the LSO and MNTB and apoptosis in the VCN and MNTB neurons, the balance at the LSO between excitatory input from the VCN and inhibitory input from the MNTB would be altered in the NB-2 KO mouse. The imbalance would potentially affect the integration of binaural sensory information at the LSO. Most of the current mouse models for impaired auditory function were generated by impairing the function of the cochlea or the peripheral auditory system (Avraham, 2003). In contrast, few mouse

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models with auditory processing disorders in the central nervous system have been reported, and thus the neuropathological mechanisms underlying these disorders remain elusive (Liu, 2006). In this respect, the NB-2 KO mouse provides a unique model for studying the mechanism of binaural pathways. In summary, we have demonstrated that NB-2 is involved in the final stage of auditory brainstem development in the mouse and that NB-2 deficiency causes significant deficits in pathway formation, leading to abnormal responses to auditory stimuli in the adult. Acknowledgments We are greatly indebted to Mr. Takeshi Hayakawa (Bio Research Center Co., Ltd.) for measurement of the ABR. We also thank to Dr. Masaki Inagaki in Aichi Cancer Center and Dr. Yasuko Tomono in Shigei Medical Research Institute for indispensable advice on monoclonal antibody production in rat. This work was partly supported by a Grantin-Aid for Scientific Research (B) (# 18300120) from the Japan Society for the Promotion of Science. References Ashley, J., Packard, M., Ataman, B., Budnik, V., 2005. Fasciclin II signals new synapse formation through amyloid precursor protein and the scaffolding protein dX11/ Mint. J. Neurosci. 25, 5943–5955. Avraham, K.B., 2003. Mouse models for deafness: lessons for the human inner ear and hearing loss. Ear Hear. 24, 332–341. Blaesse, P., Ehrhardt, S., Friauf, E., 2005. Developmental pattern of three vesicular glutamate transporters in the rat superior olivary complex. Cell Tissue Res. 320, 33–50. Caspary, D.M., Ling, L., Turner, J.G., Hughes, L.F., 2008. Inhibitory neurotransmission, plasticity and aging in the mammalian central auditory system. J. Exp. Biol. 211, 1781–1791. Eggermont, J.J., 1985. Evoked potentials as indicators of auditory maturation. Acta Oto-Laryngol., Suppl. 421, 41–47. Fullerton, B.C., Kiang, N.Y., 1990. The effect of brainstem lesions on brainstem auditory evoked potentials in the cat. Hear. Res. 49, 363–390. Hoe, H.S., Fu, Z., Makarova, A., Lee, J.Y., Lu, C., Feng, L., Pajoohesh-Ganji, A., Matsuoka, Y., Hyman, B.T., Ehlers, M.D., Vicini, S., Pak, D.T.S., Rebeck, G.W., 2009. The effects of amyloid precursor protein on postsynaptic composition and activity. J. Biol. Chem. 284, 8495–8506. Hoffpauir, B.K., Grimes, J.L., Mathers, P.H., Spirou, G.A., 2006. Synaptogenesis of the calyx of Held: rapid onset of function and one-to-one morphological innervation. J. Neurosci. 26, 5511–5523. Irvine, D.R.F., 1986. A review of the structure and function of auditory brainstem processing mechanisms. In: Ottoson, D. (Ed.), Sensory Physiology. Springer Verlag, Berlin, pp. 1–279. Kandler, K., Friauf, E., 1993. Pre and postnatal development of efferent connections of the cochlear nucleus in the rat. J. Comp. Neurol. 328, 161–184. Keith, R.W., Garza-Holquin, Y., Smolak, L., Pensak, M.L., 1987. Acoustic reflex dynamics and auditory brain stem responses in multiple sclerosis. Am. J. Otol. 8, 406–413. Kil, J., Kageyama, G.H., Semple, M.N., kitzes, L.M., 1995. Development of ventral cochlear nucleus projections to the superior olivary complex in gerbil. J. Comp. Neurol. 353, 317–340.

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