Five different animal experiments were performed in the present study, including (1) afferent projections of the ABVN in rats by using cholera toxin subunit B (CTB) tracing technique, (2) ta-VNS on t seizures in awake rats, (3) ta-VNS on electroencephalography (EEG) and the firing of the NTS neurons in anaesthetized rats, (4) ta-VNS on the field potentials (FPs) of the primary somatosensory in anaesthetized rats, and (5) the effect of reversible cooling the NTS neurons on epileptiform activity in EEG traces in anaesthetized rats. ta-VNS, transcutaneous auricular non-vagus nerve stimulation (tan-VNS), or vagus nerve stimulation (VNS) were performed in experiment (2) – (5) as needed to observe the anticonvulsant efficacy of the ta-VNS. The detailed experimental procedures are as follows.
Animals
Totally ninety four adult male healthy rats, Sprague Dawley, weighing 250–320 g, were used. Rats feeding with normal diet were housed in a room maintained at 24±1°C and illuminated for 12 hours (07:00 to 19:00) every day. Food and water were freely available. Rats were allowed to adapt to the environment for 7 days prior to the experiment. Epileptiform activity was evoked by intraperitoneal injection of pentylenetetrazol (PTZ, 60 mg/kg; Sigma-Aldrich, St. Louis, MO, U.S.A.). For anaesthesia, 10% urethane (1.2 g/kg, via intraperitoneal route) was initiated. Additional sodium pentobarbital was administered to prolong the anaesthetic state. Animals were euthanized by urethane over-dose at the end of the study. All studies were approved by the Institutional Animal Care and Use Committee of China Academy of Chinese Medical Sciences and were in accordance with National Institutes of Health guidelines.
Tracing study on central projections of primary afferent fibers of the ABVN
Microinjection was carried out on 9 rats. Under anaesthesia, microsyringe contained 1% CTB (List Biological Labs, Campbell, CA, USA) solution was inserted subcutaneously into the junction of cavity of the auricular concha and postero-inferior wall of the external acoustic meatus for a depth of 2–3 mm along the auricular surface, and a total of 3 μl 1% CTB was injected slowly. After injection, the needle was retained about one min and then withdrawn carefully. On the third surviving day, the rats were deeply anaesthetized with ether (Beijing Chemical Plant, Beijing, China) and transcardially perfused with 100 ml of 0.9% saline and immediately followed by 300 ml of 4% paraformaldehyde in 0.1M phosphate buffered solution (PB, pH 7.4).
The brain stem, cervical spinal cord and cervical ganglia were dissected out and stored in 30% sucrose PB at 4°C and allowed to sink. Serial sections of brain stem, cervical spinal cord and cervical ganglia were cut at a thickness of 40 μm on a freezing microtome (MICROM, HM 400 R, Walldorf, Germany) and collected in 0.1M PB. The sections were processed for immunofluorescence staining. In brief, sections were incubated in a blocking solution containing 3% normal rabbit serum and 0.3% Triton X-100 in 0.1M PB for 1 hr, then transferred to goat anti-CTB (List Biological Labs) at a dilution of 1:1000 in 0.1M PB containing 1% rabbit serum and 0.3% Triton X-100 for overnight at 4°C. On the following day, after washing three times with 0.1M PB, sections were exposed to rabbit anti-goat Alexa 488 secondary antibody (1:500; Molecular Probes, Eugene, OR, USA) for 2 hr and then washed with 0.1M PB. Slides were coverslipped using Immu-mount (Thermo Shandon, Pittsburgh, USA) to improve visualization of labeling. The anatomical structure of tissue sections was determined cytoarchitecturally based on The Rat Brain in Stereotaxic Coordinates (Paxinos and Watson, 1998). The tissue samples were observed and recorded with a fluorescent microscope (Y-IDP; Nikon Co., Tokyo, Japan) equipped with a digital camera (DMX1200C; Nikon, Japan). Digital images were finally processed with Adobe Photoshop CS2 (Adobe Systems, San Jose, CA, USA).
Stimulations
Bipolar silver slice electrodes (diameter of 1.5 mm) being attached to the skin with adhesive tape were introduced for ta-VNS or tan-VNS. For ta-VNS, the cathode of the electrodes was placed at the junction of cavity of the auricular concha and postero-inferior wall of the external acoustic meatus, the anode was placed at the cymba of auricular concha. For tan-VNS, the electrodes were placed on the exterior margin of the auricle outside the ABVN distribution (See Figure 1). There was 3 mm distance between the two stimulation electrodes. Operative procedure of VNS was performed as described by Dorr and Debonnel [11]. For stimulations, the electrode leads were connected with stimulator (SEN-7203 Nihon Kohden, Japan). Stimulation parameters were selected as: frequency, 20 Hz; pulse width, 0.5 ms; strength, 1.0 mA.
Observation on epileptic behaviour
Thirty awake rats were divided into three groups: control group (n=10), tan-VNS group (n=10), ta-VNS group (n=10). Rats in the control group were treated with intraperitoneal injection of PTZ. Rats in the tan-VNS group were firstly treated with tan-VNS for 30 min, then treated with intraperitoneal injection of PTZ. Rats in the ta-VNS group were firstly treated with ta-VNS for 30 min, then treated with intraperitoneal injection of PTZ. After intraperitoneal injection of PTZ, the latency of the first grand mal and the scores of epileptic seizures in the rats were observed within 30 min. Epileptic behaviours were scored according to the standard of Racine′s scale [12]. The observers of behavioral seizures were blinded to the treatment.
Simultaneous recording of EEG and the extracellular discharges of the NTS neurons in anaesthetized rats
Thirty one anesthetized rats were fixed on the stereotaxic apparatus with the incisor bar placed 3.3 mm below the interaural line. After craniotomies, bipolar silver globe electrodes (diameter of 1 mm) were placed over the dura in two holes (distance to the bregma, AP: ±1.0 mm, ML: 2.0 mm) respectively for the recording of epidural EEG. EEG signals were amplified by Amplifier WPI (World Precision Instrument, Sarasota, USA) with low frequency filter at 1 Hz and high frequency filter at 100 Hz. The extracellular discharge signals of the NTS neurons (distance to the bregma, AP: -11.3 ~ −14.3 mm; ML: 0 ~ 1.3 mm, DV: 4 ~ 7 mm) were recorded by glass microelectrodes (10-20MΩ, pulled by Narishige PE-2 vertical puller from a filamented glass) which were backfilled with 2% pontamine sky blue. Firings of the NTS neurons recorded from the glass electrodes were fed through an Xcel-3 microelectrode amplifier (FHC, Bowdoin, USA) with low frequency filter at 1 Hz and high frequency filter at 15 KHz. Both signals were captured online and analyzed offline using the CED 1401-plus data acquisition system and the Spike 2 package (Cambridge Electronic Devices, Cambridge, UK). After recording, the site was histological fixed for locating recording site. Data out of the site of the NTS were deleted.
In each rat, signals at baseline were recorded for five min firstly. Then the rat was treated with intraperitoneal injection of PTZ. Signals after PTZ were recorded for five min. After PTZ, tan-VNS, ta-VNS and VNS were randomized to stimulate in the rats for 30 s respectively. There was an interval of 30 min before beginning the next stimulation.
Microelectrode array recording of FPs in primary somatosensory in anaesthetized rats
The array with sixteen microelectrodes were implanted into the somatosensory cortices for use in recording FPs after craniotomy in 12 rats after anaesthesia. After being inserted in the correct position, the microelectrodes were cemented to skull screws by use of dental cement. FPs were collected by using Cerebus™ 5.0 Data Acquisition System at a sampling rate of 1000 Hz and a bandpass at 1–100 Hz, and were analyzed by Spike 2 package.
Firstly the rats were treated with intraperitoneal injection of PTZ. After PTZ, tan-VNS, ta-VNS were randomized to stimulate in the rats for 30 s respectively. There was an interval of 30 min before beginning the next stimulation.
Reversible cooling the NTS neurons on the EEG changes in anaesthetized rats
Twelve anaesthetized rats were fixed on the stereotaxic apparatus. An occipital craniotomy was performed in order to gain access to the dorsal surface of the medulla oblongata. A portion of the cerebellum was aspirated so that the dorsal surface of the medulla could be clearly visualized 3 mm caudal and 3 mm rostral to the obex. The bottom of a V-shaped glass tube was gently placed on the region equivalent to the area of the NTS around the obex. In order to keep the function of the NTS neurons reversible, –4°C ethylene glycol (≥98% radiochemical purity basis, PLC, aqueous solution, Sigma) was injected through the V-shaped glass tube to cool the NTS neurons physically.
There were steps for this experiment. The rats were treated with following steps including (a) intraperitoneal injection of PTZ, (b) ta-VNS for 30 s, (c) cooling of the NTS, (d) ta-VNS for 30 s during cooling of the NTS, (e) removing cooling of the NTS, (f) ta-VNS for 30 s. In the whole experiment, the EEG signals of the rats were recorded continuously.
Data analysis
In EEG tracings or FPs tracings, an epileptiform activity was shown as a highly synchronous and large-amplitude activity at least 3 times the amplitude of baseline. The firing rate of the NTS neurons was calculated in 30 s. Data were analysed with SPSS 10.0 program (SPSS Inc. Chicago, IL). Values are presented as mean ± SEM. Kolmogorov-Smirnof test was used to evaluate if groups fit normal distributions. Normally distributed groups were analysed by parametric tests. Comparison between two groups was analysed by Independent-Samples T test. Comparison between three groups was analysed by One-Way ANOVA followed by LSD or Dunnett's T3 post-hoc test. Nonnormally distributed groups were analysed by Mann–Whitney test. P < 0.05 was considered significant.