The present study was approved by the Ethics Committee of the Department of Otolaryngology, Beijing Military General Hospital (Beijing, China). C57BL/6 mice (postnatal 12–14 days; The Jackson Laboratory, Bar Harbor, ME, USA) were anesthetized with subcutaneous injections of a mixture of ketamine (50 mg/kg) and xylazine (50 mg/kg) administered in the abdominal area. The mice were decapitated, and the cortex and cerebellum were removed to expose the brainstem. The brainstem was then quickly separated and placed in a glass petri dish filled with ice-cold artificial cerebrospinal fluid (ACSF), containing 130 mM NaCl, 3 mM KCl, 1.25 mM KH₂PO₄, 20 mM NaHCO₃, 10 mM glucose, 2.5 mM CaCl₂ and 1.3 mM MgSO₄. A vibratory microtome (Vibratome; Oxford Instruments Microspec, San Mateo, CA, USA) was used to cut 200-μm trans-strial slices, which were maintained in ACSF supplemented with 5% CO₂ at 30°C.

The in vitro whole-cell current clamp electrophysiology was conducted on a recording chamber mounted on an Olympus BX51W1 upright microscope (Olympus Corporation, Tokyo, Japan) with a 63X, 0.8 N.A. water immersion objective. The recording was conducted by a Multiclamp 700A amplifier (Axon Instruments, Foster City, CA, USA), filtered at 5 KHz and digitized at 50 KHz with a 12-bit A/D digitizer (National Instruments Corporation, Austin, TX, USA). The brain slices were maintained in the recording chamber with continuous incubation with ACSF (2–4 ml/min, 95% O₂ and 5% CO₂ to maintain a pH level of 7.1–7.5). The recording pipette solution contained 120 mm potassium gluconate, 20 mm KCl, 2 mm sodium phosphocreatine, 4 mm MgATP, 10 mm HEPES, 0.3 mm NaGTP and 1.1 mm EGTA, maintained at pH 7.2 with 10 mm KOH. For the pharmacological ion-channel antagonists, 100 μM CdCl₂ was used to block the voltage-gated Ca²⁺ channels and 100 nm TTX to block the fast-inactivating sodium channels.

For western blotting analysis, the brain slices of cochlea nucleus were further micro-dissected under the microscope to separate the superficial layer, mainly consisting of CWCs, from the molecular layer, mainly constituting pyramidal neurons. After being quickly frozen on dry ice, RIPA buffer, containing 150 mm NaCl, 50 mm Tris, 1% Triton X-100, 0.1% sodium dodecyl sulfate and 1% Nadeoxycholate (pH 7.4) was used to collect brain tissue. A Bio-Rad protein assay (Bio-Rad Laboratories, Hercules, CA, USA) was then applied to equal the protein concentrations between the two types of samples. The protein lysates were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, followed by transfer onto nitrocellulose membranes (Amersham Biosciences). The blocking medium was phosphate-buffered saline (PBS) containing 0.2% Tween-20 and 5% non-fat dry milk. The lysates were then incubated with rabbit anti-Nav1.9/NaN primary antibody (1:1,000; Alomone Laboratories Ltd., Jerusalem, Israel) and goat anti-rabbit horseradish peroxidase-labeled secondary antibody, as detected by X-ray film.

The superficial layer of the cochlear nucleus slices were dissociated and plated in 6-well plates. After 1 h, the dissociated cells were fixed with 4% paraformaldehyde in 10 mm phosphate buffer (PB) at room temperature for 1 h. Next, the cells were incubated for 1 h in 0.05% Triton X-100 and 10% goat serum in PBS at room temperature, followed by incubation with primary antibody (rabbit anti-Nav1.9/NaN; 1:100) at 4°C overnight. Following three washes with PBS, the cells were incubated with polyclonal goat-anti-rabbit IgG secondary antibody (Alexa 488; 1:500; Invitrogen Life Technologies, Carlsbad, CA, USA) for 2 h at room temperature. The bottoms of the plates were then mounted with glass cover slips to prevent detachment, then imaged with a confocal microscopy (Olympus Corporation).

Mouse Nav1.9 (SCN11A) small interfering (si)RNA and scrambled siRNA (negative control) were purchased from Integrated DNA Technologies, Inc. (Coralville, IL, USA). Cochlear nucleus slices were transfected with SCN11A-siRNA (100 nm) or the scrambled siRNA using GeneSilencer (Genlantis, San Diego, CA, USA) according to the manufacturer’s instructions. Following transfection, the slices were incubated in a petri dish containing Dulbecco’s modified Eagle’s medium, 10% fetal bovine serum, N2 and B27 supplements (Invitrogen Life Technologies) at 37°C in 5% CO₂, overnight prior to electrophysiology.

Student’s t test was performed to determine significance, using SPSS version 11.0 software (SPSS Inc., Chicago, IL, USA). P<0.05 was considered to indicate a statistically significant difference.

In vitro whole-cell current-clamp recordings were conducted on 122 neurons on the superficial layer of the cochlear nucleus slides. The cells were initially held with holding potentials of ~−70 mV and 150 msec of current injections were used to generate suprathreshold responses from the cells. Among them, 84 of the recorded neurons demonstrated characteristic suprathreshold responses of CWCs, which were CAPs, consisting of trains of SAPs superimposed on slow depolarizations (Fig. 1A).

In order to identify the intrinsic sub-types of the ion channels contributing to CAPs in CWCs, short-stimuli current clamp recordings (5 msec current injections) were then utilized, along with pharmacological reagents, to block specific ion channels. The results demonstrated that under control conditions, the suprathreshold current injection elicited CAPs consisting of two SAPs on a slow-rising depolarization (Fig. 1B, left). Notably, while the voltage-gated calcium channel antagonist Cd²⁺ (100 μM) was applied in the bath medium of ACSF, the blockage of Ca²⁺ currents did not eliminate the slow depolarization. Instead it resulted in a decreased slow depolarization with a SAP superimposed on it (Fig. 1B, middle). Further application of TTX (100 nm), the antagonist of fast-inactivating sodium channels, blocked SAPs but did not block the slow depolarization.

Therefore, the results demonstrated that ionic currents, other than calcium currents and TTX-sensitive fast-inactivating sodium currents, contributed to the slow depolarizations of CAPs in CWCs.

Other experimental methods were then utilized to determine the identity of the ion channels contributing to the signature firing pattern of CAP in CWCs.

Following micro-array screening of ion channel expression in the cochlear nucleus, it was noted that Nav1.9, a TTX-resistant slow-inactivating sodium channel was likely to be expressed in the dorsal cochlear nucleus. Then, western blot analysis was used to confirm whether Nav1.9 was expressed in the dorsal cochlear nucleus or even CWCs. Following micro-dissection, the superficial layer, which mainly consisted of CWCs from the dorsal cochlear nucleus, was further separated from the fusiform layer (which mainly consisted of principal pyramidal neurons) on the cochlear nucleus brainstem slides. The two types of tissues then underwent western blot analysis using an antibody against Nav1.9. The results demonstrated that, as compared with the fusiform layer, the superficial layer had significantly stronger reaction to the Nav1.9 antibody (Fig. 2A), suggesting that the TTX-resistant slow-inactivating Nav1.9 Na⁺ channel was highly likely to be expressed in CWCs, the major cellular population in the superficial layer of the dorsal cochlear nucleus.

The western blot analysis result was also confirmed by immunohistochemistry. The superficial layer was dissociated with trituration and the dissociated cells were plated on 6-well plates. After the application of a Nav1.9 primary antibody followed by a fluorescent secondary antibody, positive staining was observed in a number of the cells (Fig. 2B). Based on morphological characterizations, those Nav1.9-positive cells had cell body sizes of ~20 μM and their dendrites branched at wide angles or even curved back toward the somas, and were therefore positively identified as CWCs from the dorsal cochlear nucleus (Fig. 2B) (12).

Finally, the functional role of Nav1.9 in contributing to the ionic properties in CWCs was examined by a loss-of-function siRNA genetic silencing assay. Brainstem slices containing dorsal cochlear nucleus were treated with either Nav1.9 siRNA (SCN11A-siRNA, 100 nm), or a non-specific control siRNA (scramble-siRNA, 100 nm). Twenty-four hours later, whole-cell current-clamp recordings were conducted on the neurons in the superficial layer of the dorsal cochlear nucleus with the addition of Cd²⁺ in the bath medium to block voltage-gated Ca²⁺ channels.

Subesquent to genetically silencing Nav1.9 in the cochlear nucleus, it was identified that the majority of the neurons from superficial layer, presumably CWCs, responded to suprathreshold current stimuli with SAPs, not CAPs (Fig. 3A). The statistical analysis demonstrated that 87±4% of the recorded CWCs fired complex spikes in the brainstem slices treated with non-specific scramble-siRNA, whereas only 11±6% of the neurons fired complex spikes in the slices treated with Nav1.9 siRNA (P<0.05; Fig. 3B). Therefore, the present results strongly suggest that Nav1.9 functionally contributed to the generation of CAPs in CWCs in the dorsal cochlear nucleus.