All experimental protocols procedures were approved by the City University Committee on Use and Care of Animals, and the Department of Health Hong Kong. All chemicals were purchased from Sigma-Aldrich (St Louis, MO). Experiments were performed on adult male Sprague–Dawley rats (250–300 g). For surgical preparations, rats were anesthetized with a mixture of xylazine and ketamine according to the protocol described in our previous publication.
Conditioned place avoidance (CPA)
Place conditioning apparatus consisted of three wooden compartments. One conditioning compartment had horizontal stripes on the walls and an odor of 1.0% acetic acid, whereas the other had vertical stripes and standardized cinnamon scent associated with it. Walls of uniform color characterized and no distinctive odor characterized the neutral compartment. The experimental process consists of three distinct sessions: a pre-conditioning session (days 1), conditioning session (day 2–4), and post-conditioning session (e.g., test days, 1, 3, 5 and 7 days after conditioning day) as we described previously . On the first day, rats were individually placed in the neutral compartment and were allowed to explore the two conditioning compartments.
Pre-conditioning day (day 1)
On day 1, the entrance connected to each compartment was opened. Rat was allowed to move freely throughout the entire apparatus (i.e., all three compartments) for 20 min. The times spent by the rat in each compartment were recorded. Animal spending more than an 80% (time spent >16 min) or less than 20% (time spent < 4 min) of the total time in a chamber were eliminated from further testing (approximately 15% of total animal).
Conditioning days (days 2–4)
The conditioning phase of all experiments consisted of 3 days. In the morning, rats received nothing, and were randomly confined with one of the compartment for 45 min. In the afternoon, rats received treatment being paired with CRD 40 mm Hg or CRD 0 mm Hg in the other conditioning compartment for 45 min. A polyethylene tube (i.d., 1.67 mm) attached to a balloon (length, 40 cm) lightly coated with a surgical lubricant was placed in the colon and secured to the base of the tail. Colorectal distension (40 mm Hg) was produced by rapidly injecting saline into the colonic balloon over 1 second and maintaining the distension for 30 seconds with 3-min interval, and repeated five times. The 0 mm Hg CRD was served as sham treatment.
In separate groups of rats the effects of s.c. injection of U69,593 (a ĸ-opiofid receptor agonist) or s.c. vehicle were paired with a distinct compartment in a place conditioning apparatus.
Post-conditioning days (1, 3, 5 and 7 days after conditioning day, test days)
The same trial was performed as pre-conditioning session (day 1). Each rat was allowed to move freely throughout the three compartments for 20 min with no aversive stimulus (CRD) presented. The time spent in each compartment was recorded.
Intravenous infusion of CCK-8
After rats were anesthetized with xylazine and ketamine (13 and 87 mg/kg body wt, respectively), a 2-cm-long heck incision was made, a polyethylene catheter (PE-10; Clay-Adams PE 10,, Becton Dickinson, Sparks, MD) was inserted into the external jugular vein and routed through subcutaneously to the back of the neck for IV infusion using a syringe-driven pump. The rats were allowed to recover; the CCK studies were performed 5 days after surgery. In our previous works we have clarified that intravenous infusion of CCK-8 at dose of 40 pmol/kg/h produced plasma CCK concentration mimic the postprandial plasma CCK levels . Therefore, in the present studies, in a group of rats, CCK-8 (40 pmol/kg/h) was infused for 15 minutes (a 250 g rat received a dose of around 2.5 pmol of CCK-8) immediately following conditioning training. The CPA studies were conducted as described above.
Intra-duodenal perfusion of 5% peptone
After rats were anesthetized with xylazine and ketamine, through a midline incision, a polyethylene cannula (Clay-Adams PE-50) was placed into the duodenum slightly above the sphincter of Oddi, and routed through the abdominal wall and subcutaneously to the back of the neck for intra-duodenal perfusion. The rats were allowed to recover; studies were performed 5 days after surgery. Immediately following conditioning training, rats received an intra-duodenal perfusion of 3 ml phosphate-buffered saline or 3 ml 5% peptone (mol wt<1000, pH 6.0, osmolality 300 mOsm/liter) for 15 min using a syringe-driven pump. The 5% peptone test solution was prepared by ultrafiltration using an Amicon membrane (YM1) and the filtrate which only contained peptides with mol wt <1000 was used as a test solution. Luminal perfusion peptone has been shown to release CCK by stimulates CCK-releasing peptide secretion [16, 35].
Perivagal application of capsaicin
To investigate the role of vagal afferent pathway in the mediation of CCK's action, we examined the effects of perivagal application of capsaicin as we described previously [20, 23, 24]. Before surgery, atropine (0.5 mg/kg ip) was administered to reduce the acute effects of capsaicin on the cardiovascular and respiratory system. After rats were anesthetized with xylazine and ketamine, a 3-cm-long midline laparotomy incision was made through the abdominal wall. The esophagus and the abdominal vagal trunks were exposed and a piece of parafilm lamina was placed beneath them to minimize the spread of capsaicin to surrounding tissues. A cotton pledget soaked in capsaicin solution (1mg of capsaicin dissolved in 1ml of vehicle (10% Tween 80 in olive oil)) was placed around the esophagus for 30min. Capsaicin drops (0.1 ml per rat) were applied every 5 min to keep the cotton moist. The surgical procedure for the control animals was identical except that only the vehicle for capsaicin (10% Tween 80 in olive oil) was perivagally applied. The area was then thoroughly rinsed with saline and dried with sterile swabs, and stitching was performed to help to close the wounds. The CRD-CPA studies combined with administration of CCK were performed 5 days after perivagal application of capsaicin. Rats were checked for normal eye wiping movement, which indicates that of perivagal application of capsaicin has no systemic effect. To demonstrate that perivagal application of capsaicin was effective in ablating capsaicin sensitive vagal afferent fibers, in our previous studies , we performed retrograde tracing studies. An aqueous suspension of fluorescent dye, True blue was injected into the anterior wall of the stomach. Animals were killed 6 days after injection for localization of the fluorescent dye. The nodose ganglia were surgically removed; substance P immunohistochemistry studies were performed for the localization of substance P. We have shown that most of the dye was found in vagal afferent neurons containing substance P immunoreactivity. In the rats treated with perivagal capsaicin, there were no True blue-labeled cells in the nodose ganglia , which suggest that capsaicin treatment in this study was effective in ablating vagal sensory afferent function. Further, our previous electrophysiological studies have demonstrated that this treatment completely abolished the CCK [23, 24], secretin-, and serotonin-elicited [18, 36] vagal afferent neuronal response at physiological concentrations. Perivagal application of capsaicin also suppressed pancreatic secretion responses induced by CCK [20, 24].
Effects of CCK-A-receptor antagonist CR-1409
A polyethylene catheter (PE-10) was inserted into the external jugular vein as described above. It has been shown that in the anesthetized rat, the peptide antagonist CR-1409 abolished cerulein-stimulated pancreatic secretion in a dose-dependent manner. At a dose of 10 mg/kg, CR-1409 abolished pancreatic response to a near-maximum dose of cerulein , and blocked the vagal afferent responses to endogenous CCK stimulation . In current studies, we examined the effects of the CCK-A-receptor antagonist CR-1409 (10 mg/kg intravenous bolus injection, dissolved in 0.005 N NaOH). CR-1409 was injected before administration of CCK-8 or luminal perfusion of peptone.
Electrophysiological recording of ACC neurons
Detailed recording procedures have been described in our previous publications [4, 26]. Briefly, anesthetized rat was place in a stereotaxic frame, an crania opening was made 1.0–5.0 mm anterior to bregma and 0.1–2.0 mm lateral to midline to record neurons in the ACC. Glass microelectrodes with tip diameters of 0.08 μm and 20–40 MΩ impedance were filled with neurobiotin and lowered into the rostral ACC by a micromanipulator (coordinates: 1.5–3.8 mm anterior to bregma, 0.3–1.0 mm lateral to midline, 1.5–3.5 mm ventral to brain surface). The rostral ACC, as defined by Vogt and Peters , is the area corresponding to perigenual Brodmann area 24b, portions of perigenual 24a, and caudodorsal area 32. After penetrating the surface of the cortex, the recording electrode was advanced until the spontaneous activity of a single unit could be accurately discriminated from the background neuronal noise. The signals were amplified by a high-input impedance preamplifier, displayed, and stored on a personal computer.
ACC neuronal activity in response to CRD
ACC neuronal spontaneous discharge was monitored for 2 min to confirm the stability of the basal firing frequency. The basal firing rate was assessed over 30 s to quantify the resting discharge in both control group and the rats infused with CCK-8. Every neuron isolated on the basis of spontaneous activity was studied to determine its response to CRD. The 50 mmHg CRD was produced by rapidly injecting saline into the balloon over 1 s and maintaining the distension for 30 s. A neuron was deemed responsive to CRD if its spike firing rate increased or decreased at least 10% from its pre-distension baseline activity. Neuronal discharge rates were measured 30 s before, 30 s during, and 120 s after CRD, with 5-min intervals in between, and evaluated on a time histogram (5-s bin width). In the control group, neurons responding to 50 mmHg CRD were tested twice to make sure the responses were consistent and repeatable . In CCK-8 treatment group, after characterizing the CRD-excited ACC neurons, steady-state basal activity was recorded; then, each neuron was further tested in response to CRD with simultaneously infusion of saline and CCK-8, respectively.
Labeling and histological identification of recording sites: Juxtacellular injection
On completion of the experiment, recorded neurons were labeled by injecting them with neurobiotin by the technique of juxtacellular iontophoresis as we described previously .
Visceromotor response (VMR) to colorectal distension (CRD)
To clarify whether administration of CCK-8 or intra-duodenal perfusion of peptone alters the visceral pain responses we measure visceral pain in animals based on brainstem reflexes, which have been described as “pseudoaffective” responses . Details of this protocol were described in our previous publications [2, 3, 7]. Briefly, teflon-coated, 32-gauge stainless steel wires were implanted into the external oblique pelvic muscles to monitor the number of abdominal muscle contractions. 5 days after surgery graded-pressure CRD (0–20–40–60 mm Hg) was produced by rapidly injecting saline into the colonic balloon over 1 second and maintaining the distention for 20 seconds to establish stimulus response curves. The results of electromyography were quantified by calculating the area under the curve (AUC), which is the sum of all recorded data points multiplied by the sample interval (in seconds) after baseline subtraction.
Statistical comparisons of the CPA data and VMR data among different treatment groups were made using two-way repeated-measures ANOVA followed by multiple comparisons adjusted by the Bonferroni test. For ACC neuronal firing, single neuronal responses were examined using Datapac 2000 (RUN Technologies, Mission Viejo, CA, USA). The prestimulus discharge frequency was assessed for 30s to quantify the resting discharge. The discharge frequency during CRD was also measured for 30s. The mean and standard deviation of ACC neuronal firing during the 30-s control period was compared with the activity after CRD. Data of spontaneous firing and firing rate to 50 mmHg CRD with saline and CCK-8 infusion were evaluated using Student’s t-test. Results were expressed as means ± SE. P < 0.05 was considered statistically significant.