Effect of Auditory Stimulation on Parasympathetic Nerve Activity in Urethane-anesthetized Rats

Materials and Methods

Animals. Male Wistar rats weighing 300-400 g were used. Animals were housed in a room maintained at 24±1°C and illuminated for 12 h every day. Food (MF; Oriental Yeast Co., Tokyo) and water were freely available. Rats were adapted to the environment for at least 1 week prior to the experiment. All animal care and handling procedures were approved by the Institutional Animal Care and Use Committee of the Institute for Protein Research, Osaka University.

Electro-physiological experiment. General preparations were performed as described previously (10). Briefly, under anesthesia (1 g/kg urethane, given intraperitoneally), a polyethylene catheter was inserted into the right femoral vein for intravenous injection. The rat was then cannulated intratracheally, while body temperature was maintained at 37.0±0.5°C using a heating pad. To record GVNA, the distal ends of the gastric branch of the ventral subdiaphragmatic vagal nerve was ligated and hooked to a pair of silver wire electrodes. The recording electrodes were immersed in a mixture of warm petroleum jelly and liquid paraffin oil to prevent dehydration and for electrical insulation. The rat was allowed to stabilize for 30-60 min after the nerve was placed on the recording electrodes. Electrical changes in GVNA were amplified 2000-5000 times with a band path of 100 to 1000 kHz, and monitored by an oscilloscope. Raw data of the nerve activity was converted to standard pulses by a window discriminator, which separated discharge from electrical background noise which remained post mortem. Both the discharge rates and the neurogram were sampled with a Power-Lab analog-to-digital converter for recording and data analysis on a computer. Data were obtained as described previously (10). Two needle electrodes were placed under the skin at the right arm and left leg to record an electrocardiogram (ECG). The ECG signal was amplified with a bioelectric amplifier (AB-620G, Nihon Kohden, Japan). The ECG was monitored with an oscilloscope, sampled with the Power-Lab, and stored on a hard disk for off-line analysis to calculate heart rate (HR).

For auditory stimulation, white noise (WN) and music (TM: “Träumerei” from Kinderszenen Op.15-7, R. Schumann) were each adjusted to average sound levels of 50 dB. Because WN at 60 dB but not 50 dB elevated RSNA, BP and HR in preliminary experiments, 50 dB was used as the loudness for WN and music in the experiments. The stimulation was presented for 60 min by repeating WN or TM through the earphones, which were fixed to both ears before the stabilization period. At the end of the experiment, hexamethonium chloride (10 mg/kg) was administered intravenously to ensure that postganglionic efferent parasympathetic nerve activity had been recorded.

Immunohistochemistry. Rats were randomly divided into three groups: no stimulation (NST) and auditory stimulation with WN or TM. After the experiment, the rats were perfused transcardially with 4% paraformaldehyde. Their brains were soaked in the same fixative for 24 h and in 20% sucrose for 48 h, and then frozen in OCT compound and cut in 20-μm sections using a cryostat (total of 15 sections [1 of every 6 sections]/rat) referring the atlas of Paxisons and Watsons (11). Coronal sections through the auditory cortex (AuC) were treated with an anti-c-Fos polyclonal rabbit antibody (Abcam plc, Cambridge, UK) and then with biotinylated anti-rabbit IgG (Vector Laboratories, Burlingame, CA, USA). Sections were then treated with ABC kits (Vector Laboratories) according to the manufacturer's instructions. The number of cells immunoreactive (IR) for c-Fos in the stained sections was quantified using Image J (NIH Image).

Data analysis. The GVNA data measured during each 5-min period after the start of auditory stimulation were analyzed by digital signal processing and appropriate statistical analyses. All data were expressed as means±SEM. Because of the inter-individual variability in the pre-stimulation state, percent changes from the baseline values were calculated for the GVNA. The Mann-Whitney U-test was used to compare the baseline values in the groups. Two-way ANOVA was used to compare group responses of the GVNA to auditory stimulation. To perform statistical analysis of the c-Fos data, ANOVA, followed by multiple comparisons using Dunnett's multiple range tests, was used. P<0.05 was considered statistically significant.