Animals and Chemicals.

For GeneChip analysis, 16 Beagle dogs consisting of 8 males and 8 females (7.5–10.5 kg b.wt.) were purchased from the Teaching and Practicing Farm, Institute of Agriculture, Shanghai Communication University (Shanghai, People’s Republic of China). The purchase was qualified and certified by Shanghai Laboratory Animal Management Committee [SCXK(Hu)2007-0004]. For the motion sickness test, another 12 Beagle dogs consisting of 6 males and 6 females (7.8–10.2 kg b.wt.) were purchased from Yangzhou Sifang Experimental Animal Technology Co., Ltd. (Yangzhou, People’s Republic of China). The purchase was qualified and certified by the Jiangsu Laboratory Animal Management Committee [SCXK(Su)2008-0006]. Sprague-Dawley rats with half males and half females (220–250 g b.wt.) and pregnant dams were obtained from the Experimental Animal Center of Nantong University (Nantong, People’s Republic of China). The experimenters were blinded for the grouping and genotype of the animals. All procedures used in this study were used in accordance with our institutional guidelines, which comply with international rules and policies and were approved by the Animal Care and Use Committee of Nantong University.

Common inorganic salts were purchased in the People’s Republic of China, and cell culture medium (B27, Dulbecco’s minimum essential medium, fetal bovine serum, F12, and neurobasal medium) was purchased from Gibco (Carlsbad, CA). GeneChips (GeneChip Canine Genome 2.0 Array) were purchased from Affymetrix China (Shanghai, People’s Republic of China). Tacrine was purchased from Shanghai Hebao Chemical Co., Ltd. (Shanghai, People’s Republic of China), dimenhydrinate was purchased from Shanghai Huaihai Pharmaceutical Factory (Shanghai, People’s Republic of China), and saccharin sodium was purchased from Tianjin North Food Co., Ltd. (Tianjin, People’s Republic of China). Pentobarbital sodium, chloral hydrate, glutamine, promethazine, poly-d-lysine, cytosine-β-d-arabinofuranoside, histamine dihydrochloride, dansyl chloride, mouse anti-rat and dog β-actin antibodies, and other chemicals (except those indicated elsewhere) were purchased from Sigma-Aldrich (St. Louis, MO). Rabbit anti-rat, dog HNMT, goat anti-rabbit IgG (H+L)-horseradish peroxidase (HRP), and goat anti-mouse IgG (H+L)-HRP antibodies were purchased from Bioworld China (Nanjing, People’s Republic of China).

Motion Sickness Induction via Rotatory Stimulus.

The rotatory stimulator for animal use was produced in reference to that reported by Crampton and Lucot (1985) and described in our previous study (Li et al., 2005). To induce motion sickness, dogs and rats in the stimulator were rotated in an alternate acceleration and deceleration mode. The acceleration rate was 16°/s² for a duration of 7.5 seconds, and a maximal velocity of 120°/s was reached. A duration of 2.5 seconds was used for deceleration with a rate of 48°/s². Clockwise and counterclockwise rotations were alternately repeated for 30 minutes for the dogs and 120 minutes for the rats.

Vomiting served as the index of motion sickness in the dogs. The same rotatory test was repeated 2 weeks later. After averaging the results of two tests, the dogs with a vomiting onset time of less than 30 minutes were considered susceptible to motion sickness, whereas dogs with a vomiting onset time of more than 30 minutes were considered insusceptible.

Conditioned taste aversion, as suggested by the literature (Ossenkopp, 1983; Fox and McKenna, 1988; Sutton et al., 1988; Gallo et al., 1999; Li et al., 2005), was used as the index of rat motion sickness. Before the rotatory stimulus, saccharin sodium solution (SSS; 0.15%) was provided for the rats to drink for 45 minutes when they had been deprived of water for a 24-hour period and had become familiar with this novel fluid 1 day before water deprivation through free access to 0.15% SSS in addition to tap water for another 24 hours. After a 24-hour period of rest with free access to tap water, the rats were rotated, and, after another 24-hour period of water deprivation, 0.15% SSS was then provided for 45 minutes and the SSS intake volume was measured. The same rotatory test was repeated 2 weeks later. After averaging the results of two tests, the rats with a decrease in 0.15% SSS intake of less than 15% were considered insusceptible to motion sickness, whereas the rats with a decrease in intake greater than 15% were considered susceptible. As a control, control rats of both sexes before and after the rotatory stimulus were supplied with tap water instead of 0.15% SSS for the same duration of 45 minutes after water deprivation for a 24-hour period. The protocol of the conditioned taste aversion test is shown in Fig. 1.

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Fig. 1.

Protocols of the conditioned taste aversion test in rats.

Adaptive Vestibular Training.

For adaptive vestibular training, 14 susceptible rats of both sexes were selected. Seven rats were used as controls without training, and another seven rats were subjected to 1 month of training. The training group was rotated every 2 days with the same rotatory stimulus as described above for a duration of 60 minutes.

Drug Tests on Motion Sickness.

Six susceptible dogs were selected for the drug test study, which consisted of a 6 × 6 Latin square design with one test conducted every week (i.e., two controls: dimenhydrinate 0.3125 mg/kg; and tacrine 0.1875, 0.375, and 0.75 mg/kg) and repeated for 6 weeks, and each dog was subjected to six tests in a random order for the 6-week experiment design; data from two control dogs in every experiment were averaged as one sample for statistical analysis. The rotatory stimulus was applied for a duration of 30 minutes or until vomiting occurred. Drugs were administered orally 60 minutes before rotatory stimulus, and the control dogs were given an equal volume of 10 ml of normal saline.

Forty-eight insusceptible rats were randomly allocated to four groups (i.e., control, and tacrine 0.675, 1.35, and 2.7 mg/kg). Tacrine was delivered via intraperitoneal injection 30 minutes before rotatory stimulus of 2 hours duration, and the control rats received an equal injection volume of 1 ml of normal saline per 200 g weight.

Intracerebral Microinjection.

The rats were anesthetized with 10% chloral hydrate (400 mg/kg, i.p.) and placed in a stereotaxic frame (Stoelting, Wood Dale, IL). Over a 15-minute period, 0.5 μl of promethazine solution was slowly injected into one side of the dorsal vagal complex (DVC) using a 1-μl microsyringe (Hamilton, Reno, NV) driven by an injector (Quintessential Stereotaxic Injector; Stoelting). The DVC coordinates for the injection site were 14.04 mm posterior to the bregma, 0.7 mm lateral to the midline, and a depth of 8.0 mm under the meninges (Paxinos and Watson, 2005), with a retention time of 10 minutes. The microinjection point was next to the dorsal nucleus of the vagal nerve and the nucleus of the solitary tract. Microinjection for the other side of the DVC was successively conducted in the same way. Control rats were injected with normal saline at an equal volume. Similarly, HNMT-overexpressing and HMNT-inhibitory lentiviruses (LVs) and empty-vector controls (1.5 × 10⁶ transducing units (TU)/2 μl for each side) were respectively injected into both sides of the DVC through a 10-μl microsyringe (Hamilton).

Primary Cell Culture.

Rat embryos (embryonic days 17–18) were harvested from pregnant rats anesthetized with 10% chloral hydrate (400 mg/kg, i.p.). The embryos were placed in sterile ice-cold Hank’s balanced salt solution (Gibco). The embryonic rat brains were taken out, and both sides of the cortex were carefully dissected. The tissue matrix was digested with 0.25% trypsin (Life Technologies, Carlsbad, CA) at 37°C in a shaking water bath for 6 minutes. After mechanical trituration and centrifugation, the final cell pellet was resuspended in Dulbecco’s minimum essential medium containing 10% fetal bovine serum, 10% F12, and 1% penicillin-streptomycin. The cells were seeded in 24-well poly-d-lysine–coated plates at a density of 5 × 10⁵ cells/ml. Ten hours later, the medium was replaced with neurobasal medium supplemented with 2% B27, 1% glutamine, and 1% penicillin-streptomycin. The cultures were maintained by a 50% medium substitution every 3 days. The day of plating was counted as in vitro day 0. On in vitro day 3, cytosine-β-d-arabinofuranoside (5 μmol) was used to suppress the proliferation of glial cells for 3 days, and then the regular neurobasal medium was recovered. The cell plates were put in a humidified 5% CO₂ incubator at 37°C.

Nissl Staining and Immunofluorescent Observation.

The rats were perfused with 200 ml of saline under 10% chloral hydrate (400 mg/kg, i.p.) anesthesia and then with 4% paraformaldehyde in 0.1 mol phosphate-buffered saline (pH 7.4). The rat brains were harvested and postfixed for 24 hours. The fixed brains were then protected in phosphate-buffered saline containing 30% sucrose. For Nissl staining, the brains were coronally cut into slices with a thickness of 30 μm using a cryostat (CM1900; Leica, Bensheim, Germany). For immunofluorescence, the brains were cut into slices with a thickness of 5 μm.

For Nissl staining, the brain slices were mounted with neutral balata, blotted onto slides, and processed through a series of baths in the following order: chloroform (30 minutes), acetone (15 minutes), 100% ethanol (30 seconds), 95% ethanol (30 seconds), 70% ethanol (30 seconds), distilled water (30 seconds, twice), cresyl violet (20 minutes), distilled water (30 seconds, three times), 70% ethanol (1 minutes), 95% ethanol (1 minutes), 100% ethanol (1 minutes), chloroform (5 minutes), differentiator (95% ethanol, added glacial acetic acid until the pH was 4.1, 6 minutes), 95% ethanol (2 minutes), 100% ethanol (3 minutes, twice), xylene (2 minutes), and xylene (3 minutes, twice). Afterward, the slices were coverslipped.

For immunofluorescent detection of the HNMT gene transfection, we transfected the cultured cells and the DVC with a lentiviral vector expressing green fluorescent protein (GFP). The brain slices and cultured cells were imaged through a laser confocal microscope (TCS SP8; Leica Microsystems, Wetzlar, Germany) at room temperature and processed with software LAS X version 2.0 (Leica Microsystems).

GeneChip Analysis.

The dog brains were harvested under anesthesia (3% pentobarbital sodium, 1 ml/kg, injected through the small saphenous vein of the foreleg). The medulla oblongata, pons, diencephalon, and vestibular cerebellum were isolated. RNA was prepared by pulverizing tissues in a mortar followed by the addition of TRIzol reagent (1 ml/100 mg of tissue; Invitrogen, Carlsbad, CA). The RNA quality was evaluated with denaturing gel electrophoresis. RNA was purified with a Qiagen miRNeasy Kit (Qiagen, Düsseldorf, Germany), and RNA concentration was determined using a NanoDrop ND-2000 Spectrophotometer (Thermo Scientific, Waltham, MA).

Each sample (500 ng) of total RNA was used to prepare biotinylated fragmented cDNA according to the Gene-Chip Expression Analysis Technical Manual (Revision 5; Affymetrix, Santa Clara, CA). The Affymetrix GeneChip expression arrays (Canine Genome 2.0 Array) were hybridized in an incubator at 45°C at 60 rpm for 16 hours. The arrays were washed and stained using the Fluidics Station 450 (Affymetrix) and scanned using the GeneChip Scanner 7G (Affymetrix). The analysis of gene expression data was accomplished using packages available from the Bioconductor project (www.bioconductor.org). The raw data were normalized using the robust multiarray average background-adjusted, normalized, and log-transformed summarized values to search for differentially expressed genes.

A total of 16 GeneChip arrays were used. Dog brain tissue samples from four susceptible males, seven susceptible females, or four insusceptible males were pooled respectively (i.e., one sample from the medulla oblongata, pons, diencephalon, and vestibular cerebellum was respectively used for final GeneChip array measurement). Another part of the samples from these four brain areas was obtained from one insusceptible female dog. Data from susceptible (11 dogs) and insusceptible (5 dogs) animals of both sexes were compared.

Construction of Recombinant Lentiviral Vector and Transfection to DVC.

A recombinant lentiviral vector–expressing rat HNMT-short hairpin RNA (shRNA) (Lv-Hnmt-RNAi) was constructed by Shanghai GeneChem (Shanghai, People’s Republic of China) to interfere with the expression of the HNMT gene. HNMT-shRNA was introduced into GV118-RNAi vector that carried the GFP reporter gene driven by the U6 promoter. The following three vectors were designed: LV-shRNA1-HNMT (target sequences: 5′-GGAGACATCATCTGAATAT-3′); LV-shRNA2-HNMT (5′-GCTTCCAGGCATAATAGCA-3′); and LV-shRNA3-HNMT (5′-GAGCCAAATGCCGAACAAA-3′). The suppression of HNMT mRNA expression was analyzed by quantitative real-time PCR (qRT-PCR), and HNMT protein levels were quantified using Western blot analysis. The most efficient recombinant vector was used in later experiments. GV118-RNAi vector, which produces a nontargeting sequence, 5′-TTCTCCGAACGTGTCACGT-3′, was used as the negative control (LV-shRNA-NC). The recombinant vectors and the shRNA-LVs were cotransfected into human embryonic kidney 293 T cells and the titer of recombinant lentiviruses was 6 × 10⁸ infectious units/ml. Another recombinant lentiviral vector based on the GV287-GFP vector was constructed expressing the HNMT gene (Lv-Hnmt; Shanghai GeneChem), and the titer of recombinant lentivirus was 2 × 10⁸ infectious units/ml.

Lentiviruses of 1.5 × 10⁶ TU/2 μl were used for microinjection into each side of the DVC in the in vivo experiment. Ten days after the injection of lentiviruses, the rats were subjected to rotatory stimulus and later measurement of conditioned taste aversion to 0.15% SSS. In the in vitro transfection experiment, lentiviruses were added to the medium of cultured neurons on in vitro day 7. After 10 hours, the medium was changed with neurobasal medium supplemented with 2% B27, 1% glutamine, and 1% penicillin-streptomycin. Half of the medium was changed 3 days later. After 4 days, protein was extracted from the cultured neurons for analysis.

Quantitative Real-Time PCR.

The rat brains were obtained under 10% chloral hydrate (400 mg/kg, i.p.) anesthesia, and the medulla oblongata was isolated on an ice plate. For isolation of the DVC, the rat medulla oblongata was sectioned into 300-μm-thick slices in ice-cold artificial cerebrospinal fluid using a vibratome (Campden Instruments Ltd., Loughborough, UK). According to the DVC coordinate in the brain atlas (Paxinos and Watson, 2005), the area postrema, nucleus of the solitary tract, and dorsal nucleus of vagus nerve were cautiously isolated for qRT-PCR and Western blot analysis. The dog brains were isolated as described above for GeneChip measurement.

The brain tissues isolated from the rats and dogs or cell cultures were put into TRIzol reagent (Invitrogen) and homogenized with a homogenizer, and the total RNA was then purified. Potential DNA contamination was removed with DNase. One microgram of RNA was used for first-strand cDNA synthesis through retrotranscription according to the manufacturer instructions using oligo(dT) primers and the PrimeScript RT Reagent Kit [Tiangen Biotech (Beijing) Co., Ltd, Beijing, People’s Republic of China]. The qRT-PCRs were performed using a Rotor-Gene PCR machine (RG-3000A; Corbett Research Pty. Ltd., Mortlake, NSW, Australia). The qRT-PCR procedures were as follows: a first denaturation step at 95°C for 5 seconds, followed by 40 cycles of a 95°C denaturation for 5 seconds, 60°C annealing for 20 seconds, and 72°C extension for 30 seconds. The amount of cDNA per sample was measured through a SYBR Premix Ex Taq kit (Roche, Basel, Switzerland). The progression of the PCRs was estimated through variations in the SYBR green dye fluorescence attached to double-stranded DNA. All values were normalized to the housekeeping gene β-actin. The primers used for qRT-PCR of the dog samples were β-actin (forward 5′-TGGCCGGGACCTGACTGACTACCT-3′, reverse 5′-CCCAACCCTGTTAGACCGGCAAGAC-3′) and HNMT (forward 5′-CCAGGCATAACAGCAAGGA-3′, reverse 5′-TCCCAGCCACTGTTTCCT-3′). The primers used for qRT-PCR of the rat samples were β-actin (forward 5′-TCTACATGTTCCAGTATGACTC-3′, reverse 5′-ACTCCACGACATACTCAGCACC-3′) and HNMT (forward 5′-AACAGCACCACTGGACCTCA-3′, reverse 5′-TTGCCTCAACCACTATAAAACTCA-3′).

Western Blot Analysis.

The brain tissues and cell cultures were lysed in lysis buffer containing phenylmethanesulfonyl fluoride (Beyotime, Nantong, People’s Republic of China) at 1 mmol and centrifuged at 13,400g at 4°C for 15 minutes. The supernatant protein content was measured by the bicinchoninic acid method and read on a spectrophotometer. Equal amounts of protein from each sample (40 μg/lane) were loaded on a 10% SDS-polyacrylamide gel, electrophoresed, and transferred onto polyvinylidene difluoride membranes (Millipore, Burlington, MA). The membranes were blocked with Tris-buffered saline containing 5% nonfat milk and 0.1% Tween-20 at room temperature for 2 hours and incubated at 4°C overnight with primary antibodies that were diluted in Tris-buffered saline containing 5% nonfat milk and 0.1% Tween-20 to HNMT (1:500) and β-actin (1:1000). After incubating the blots with goat anti-rabbit IgG (H+L)-HRP and goat anti-mouse IgG (H+L)-HRP (1:10,000) at room temperature for 2 hours, the immunoreactive bands were visualized using enhanced chemiluminescent agents (Pierce Biotechnology, Waltham, MA) and captured using a Tanon 5200 Multi Chemiluminescent System (Tanon, Shanghai, People’s Republic of China). The expression level of each protein was quantified using ImageJ analysis software and normalized to the β-actin value in the same lane. The same samples were repeatedly analyzed at least two times.

High-Performance Liquid Chromatography Measurement of Histamine.

High-performance liquid chromatography (HPLC) measurement of histamine content in the brain tissue was performed with reference to literature (Yamatodani et al., 1985; Altieri et al., 2016). Twenty-four rats were randomly allocated to three groups (i.e., the control, rotation, and tacrine 2.7 mg/kg groups). Tacrine was delivered via intraperitoneal injection 30 minutes before a 2-hour rotatory stimulus, and the control rats received an equal volume of normal saline (1 ml/200 g weight). Immediately after the rotatory stimulus, the rat brain was harvested by decapitation under anesthesia, and the medulla oblongata was quickly isolated on ice. The brain tissues were weighed and homogenized with a handheld homogenizer after the addition of five volumes of extraction solution (0.4 mol perchloric acid), and the supernatants were collected after centrifugation at 12,000 rpm for 20 minutes at 4°C. All solvents used for HPLC analysis were of HPLC grade. The HPLC system used was the Waters e2695 series (Waters, Milford, MA) equipped with a 2998 Photodiode Array Detector (Waters) and a Sunfire C18 column (5.0 μm; column dimension, 6 × 250 mm; Waters). Each standard solution or sample (100 μl) was mixed with 20 μl of 2 N sodium hydroxide, 30 μl of saturated sodium bicarbonate, and 200 μl of dansyl chloride solution (10 mg/ml, prepared in acetone), incubated at 40°C for 45 minutes after mixing thoroughly and then cooled down to room temperature in 10 minutes. The residual dansyl chloride was removed with the addition of 10 μl of 25% ammonia solution. Thirty minutes later, the mixture was adjusted to a volume of 1 ml with ammonium acetate (0.01 mol, pH 7.9) and acetonitrile (1:1 v/v), and filtered through a syringe filter (0.45 μm pore size). Measurement of histamine level was performed at a flow rate of 1.0 ml/min with the column temperature at 40°C. Ammonium acetate was used as solvent A, and acetonitrile as solvent B. The gradient was applied as follows: from 35% to 70% (v/v) solvent B in solvent A for 15 minutes; from 70% to 95% (v/v) solvent B in solvent A within 10 minutes; and equilibrated at 95% (v/v) solvent B in solvent A for 5 minutes before the next run. The injection volume for standard solutions and samples was 20 μl, and histamine was detected at a wavelength of 254 nm. The content of tissue histamine was expressed as nanomoles per gram of wet tissue weight (nanomoles per gram) in absolute values.

Statistical Analysis.

All data are presented as the mean ± S.D. The two-group design was analyzed with a Student’s t test. The three-or-more group design was analyzed with a one-way analysis of variance followed by the Fisher’s least significant difference test for post hoc comparisons between two groups. Differences were considered statistically significant at P < 0.05.