Heterozygous necdin-deficient mice (B6.Cg-Ndntm1ky) were purchased from RIKEN (Saitama, Japan). Ndn was maternally imprinted, and only the paternal allele was functional. Therefore, heterozygous males carrying an NDN-deleted allele were bred from wild-type (WT) females (ICR background) to generate WT (+m/+p) and heterozygotes (+m/−p) animals in which the paternal allele was deleted, leading to necdin deficiency. These animals were used in all experiments. The use of animals was approved by the Ethical Committee for Animal Research of the Buddhist Taipei Tzu Chi General Hospital (105-IACUC-017) and was accordance with the National Institutes of Health guidelines. Every effort was made to minimize the number of animals used and their suffering.
Mice were anesthetized with urethane (1.3 g/kg) via intraperitoneal injection and perfused transcardially through the left ventricle with saline, followed by 0.1 M phosphate-buffer (PB, pH 7.4) containing 4% paraformaldehyde. The brain was dissected and fixed overnight in the same fixation solution at 4°C and stored in 30% sucrose in 0.05 M PB. To evaluate the necdin expression in the CNS, serial coronal sections were cut into 50-μm-thick sections using a freezing microtome. Slices were incubated for 1 h at room temperature in phosphate-buffered saline containing 0.03% Triton X-100 (PBST), 2% bovine serum albumin, and 10% normal goat serum and then incubated overnight at 4°C in PBST containing 1/1000 dilution of rabbit antibodies against necdin (Bio Academia, Osaka, Japan) for 40 h at 4°C. After rinsing with PBST, tissue slices were incubated with the secondary antibodies for 3 h: 1/200 dilution of goat anti-rabbit IgG-conjugated Alexa Fluor 594 (Jackson ImmunoResearch, West Grove, PA, USA). After rinsing with PB, the slices were mounted with RapiClear 1.47 (SunJin Lab, Hsinchu City, Taiwan), and a cover slip was placed. Immunofluorescence images were observed using a Leica SP8 confocal microscope (Leica microsystems, Wetzlar, Germany).
Preparation of brainstem slices
The animals were anesthetized with 5% isoflurane in O2 and decapitated. The brains were rapidly exposed and chilled with ice-cold artificial cerebrospinal fluid (ACSF) with 119 mM NaCl, 2.5 mM KCl, 1.3 mM MgSO4, 26.2 mM NaHCO3, 1 mM NAH2PO4, 2.5 mM CaCl2, and 11 mM glucose, oxygenated with 95% O2 and 5% CO2 at pH 7.4. Coronal brainstem slices (300 μm) harboring LC were prepared using a vibratome (VT1000S, Leica, Wetzlar, Germany), maintained in a moist air–liquid (ACSF) interface chamber at room temperature (24°C–25°C), and allowed to recover for at least 90 min. LC was identified as a transparent, long, oval area located rostral to the floor of the 4th ventricle and beneath the superior cerebellar peduncle.
Brainstem slices were transferred to a perfusion chamber mounted on an upright microscope (BX51WI, Olympus Optical Co., Ltd., Tokyo, Japan) and continuously perfused with oxygenated ACSF at 2–3 ml min−1. Neurons were viewed using a digital camera (C10600 ORCA‐R2, Hamamatsu, Japan). Patch pipettes, pulled from borosilicate glass tubing (outer diameter, 1.5 mm; wall thickness, 0.32 mm; Warner Instruments Corp., Hamden, CT, USA), showed a resistance of 3–5 MΩ when filled with the pipetting solutions. All experiments were recorded using a patch amplifier (Multiclamp 700 B; Axon Instruments Inc., Union City, CA, USA). Signals were low pass-filtered at 2 kHz and digitized at 10 kHz using Micro 1401 + Spike2 (Cambridge Electronic Design, Cambridge, UK). For intracellular recordings, the pipette solution constituted (in mM): 131 K-gluconate, 20 KCl, 10 HEPES, 2 EGTA, 8 NaCl, 2 ATP, and 0.3 GTP (pH, 7.2–7.3; osmolarity, 300–305 mOsm). For current-clamp recordings, the bridge was balanced and recordings were only accepted if the recorded neuron showed a membrane potential (Vm) of at least –45 mV without applying a holding current and if the action potentials (APs) could overshoot 0 mV. For voltage-clamp recording, Vm was clamped to −70 mV, unless otherwise specified. For cell-attached voltage-clamp recording, spontaneous AP recordings were made at 29°C–31°C. The pipette solution was replaced by normal ACSF (24, 25). During the electrophysiological recordings, 5 mM kynurenic acid (Sigma, St Louis, MO, USA) and 0.1 mM picrotoxin (Sigma, St Louis, MO, USA) were added in bathing ACSF to block glutamatergic and GABAergic synaptic transmission. For the voltage-clamp recording of A-type K+ currents (IA), 1μM tetrodotoxin (Tocris Cookson, Bristol, UK) was mixed in calcium-free bathing ACSF to avoid contamination of the Na and Ca currents.
Evaluation of developmental reflexes
Developmental reflexes, including surface righting, bar holding, and negative geotaxis, were tested to evaluate muscle tone and physical endurance in mice. Three cohorts of male mice were used in the evaluation of developmental reflexes from postnatal day 3 to 13. The experiments were conducted according to previously reported protocols (26). To evaluate surface righting, mice pups were gently placed on their backs, and the time taken for them to turn onto their bellies was recorded. To evaluate bar holding, mice pups were lifted by the trunk and held close to a thin metal bar. The period of hanging by their front paws was recorded. To evaluate cliff avoidance, mice pups were placed on the edge of a wooden platform with their noses and forefeet over the edge. The period taken by each pup to move away from the cliff by backing up or by turning sideways was recorded.
Evaluation of respiration
The tested mice were placed in a whole-body plethysmograph (EMKA Technologies, Paris, France) for 60 min under normocapnic gas (78.5% N2, 21% O2, and 0.5% CO2) with a ventilation pump. Hypercapnic gas (72% N2, 21% O2, and 7% CO2) was applied for 20 min, followed by a period of recovery under normocapnic gas for 20 min. Apnea was manually measured and was defined as the absence of at least two inspirations (27). The ventilatory parameters, including tidal volume, minute volume, and frequency of breathing, were determined using IOX v2 (EMKA Technologies, Paris, France).
Data and statistical analyses
Statistical analyses were performed using SPSS version 19.0 for Windows (IBM, Chicago, IL, USA). All data were presented as mean and standard error of mean. To assess the significance of independent variables in WT and Ndn +m/−p mice, Student’s t test was used. The level of significance was set at P < 0.05.