All methods used in this study were approved by the University of Calgary Animal Care Committee and conform to the guidelines of the Canadian Council on Animal Care. Adult male guinea-pigs (Charles River, Montreal, Canada) weighing between 250 and 350 g, were housed in cages with soft bedding and maintained on a 12: 12 h light–dark cycle. The animals were allowed access to food and water ad libitum. Colitis was induced by intracolonic administration of 0.5 ml of TNBS (25 mg ml−1) in 25% ethanol as previously described (Linden et al. 2003). Briefly, animals were anaesthetized with halothane (induced at 4%, maintained at 2% in oxygen; MTC Pharmaceuticals, Cambridge, ON, Canada) and given an enema of TNBS through a polyethylene catheter inserted rectally whose tip was placed 7 cm proximal to the anus. Control animals remained naive until tissue collection. Previous work detected no differences in electrophysiological characteristics of colonic myenteric neurones between naive animals and animals given an enema of saline (Linden et al. 2003).
Six days after TNBS administration, animals were deeply anaesthetized with halothane and exsanguinated. The distal colon was removed, the oral end marked, and placed in Kreb's solution (mm: NaCl, 117; KCl, 4.8; CaCl2, 2.5; MgCl2, 1.2; NaHCO3, 25; NaH2PO4, 1.2; and d-glucose 11; aerated with 95% O2–5% CO2) containing nicardipine (3 μm) and scopolamine (1 μm). The segment of colon that had the most marked hyperaemia due to inflammation was opened along its mesenteric border and pinned flat, mucosa uppermost, in a dissecting dish lined with sylastic elastomer (Dow-Corning, Midland, MI, USA) that contained aerated Kreb's solution. Following tissue collection, the severity of colitis was assessed by weight change and by macroscopic colonic damage scoring that included the severity and size of the area of mucosal damage (McCafferty et al. 1997).
To prepare the tissues for recording, the mucosa was gently removed from the underlying submucosa using fine forceps from the region of distal colon that had the most severe inflammation. This region was located between 5 and 10 cm from the pelvic brim. Tissues from control animals were also taken from 5 to 10 cm proximal to the pelvic brim. Following removal of the mucosa, the preparation was then turned over and pinned out with the serosal side up. The serosa and external musculature were removed to yield a preparation of submucosa. The oral right-hand corner of the preparation was marked with a cut to aid later morphological analysis, and the preparation was transferred to a small (volume 2.5 ml) sylastic elastomer-lined recording dish and pinned flat using 80 μm diameter tungsten pins with the surface that had faced the circular muscle upwards. The recording dish was then transferred to the stage of an inverted microscope (Nikon Diaphot) and continuously superfused with aerated Kreb's solution containing nicardipine and scopolamine that had been preheated to yield a bath temperature of 36°C. The tissue was equilibrated with Kreb's solution for 1 h before recording commenced. This period was chosen to verify the viability of the preparation, and to enable any released inflammatory mediators to be washed from the organ bath.
Neurones were impaled with microelectrodes fabricated from 1 mm outer diameter borosilicate glass (World Precision Instruments, Sarasota, FL, USA) that were filled with 1% biocytin in 1 m KCl. Electrode resistances were 70–120 MΩ. Recordings of membrane potential were made using a Multiclamp 700A amplifier in current clamp mode (Axon Instruments, Foster City, CA, USA). Signals were digitized at 5–50 kHz (Digidata 1322A; Axon Instruments) and stored using PC-based data acquisition and analysis software (pCLAMP 9.2 suite, Axon Instruments). Electrophysiological variables were measured offline using Clampfit 9.2 (Axon Instruments).
Neuronal electrical properties were determined after allowing the impalements to stabilize for 5 min without applying intracellular holding current. At this time, the ability of the cell to fire an AP upon intracellular current injection was assessed. Only neurones that were able to fire an AP that overshot 0 mV and had resting membrane potentials more negative than −40 mV were included in the electrophysiological analyses.
Excitability was measured by injecting 500 ms depolarizing and hyperpolarizing current pulses whose amplitude increased in 20 pA increments. This protocol revealed input resistance, whether the neurone fired anode break APs, the number of APs at rheobase, twice rheobase, and the maximum number of APs that each neurone could fire. Synaptic inputs and antidromic APs of neurones were stimulated using bipolar silver chloride electrodes (10–50 μm tip diameter), insulated except at the tip, that were carefully placed on interganglionic nerve bundles. Stimulus pulses of 0.5 ms duration and 5–15 V intensity were delivered via a Grass SD9 stimulator (Grass Medical Instruments, Quincy, MA, USA); stimulation electrode location and stimulation intensity were adjusted to evoke maximal synaptic potential amplitudes for each neurone in both experimental groups. For analysis of fast EPSP amplitudes, neurones were hyperpolarized to −80 mV to prevent APs and to minimize changes in the electrochemical driving force, and the average amplitude of at least three fast EPSPs at −80 mV was recorded for each neurone under control conditions and following superfusion with receptor antagonists. Antagonists of ionotropic nicotinic receptors (hexamethonium, 100 μm), P2X receptors (PPADs, 30 μm) and 5-HT3 receptors (Granisetron, 1 μm) were utilized in the present work. One second trains of pulses at 20 Hz were applied to internodal strands to determine whether the impaled neurones received slow synaptic input. Slow EPSP amplitudes were measured from slow EPSPs evoked at the resting membrane potential of each neurone.
After recording, neurones were injected with biocytin using the protocol of Lomax et al. (2001). Once a neurone in a ganglion had been injected with biocytin, the ganglion was drawn and the recording electrode was moved to a fresh ganglion to avoid ambiguity of cell identity. At the end of each experiment, the tissue was fixed overnight in Zamboni's fixative (0.1 m phosphate-buffered saline (PBS) containing 2% formaldehyde plus 0.2% picric acid), and washed in three changes of PBS. The tissue was then incubated with avidin coupled to fluorescein isothiocyanate (FITC; 1: 100, Sigma Aldrich) to reveal biocytin.
Neurones were classified as AH or S neurones according to well established criteria (Bornstein et al. 1994; Wood, 1994). In accordance with previous work, only neurones with AH electrophysiological characteristics had multiaxonal Dogiel type II morphology. Likewise, S neurones had uniaxonal Dogiel type I morphology or filamentous Dogiel type III morphology (Lomax et al. 2001). In a previous study a third electrophysiological class of neurone was identified (Lomax et al. 2001). This class of neurone, called LT neurones, had similar characteristics to hyperexcitable AH neurones but did not have Dogiel type II morphology. We recorded from four LT neurones in control preparations and no LT neurones from inflamed preparations so these neurones will not be discussed further as no comparisons could be made between control and inflamed neurones.
Agonists of ligand-gated ion channels (acetylcholine [ACh], 1 mm; 5-HT, 300 μm: ATP, 1 mm) were applied to the ganglia containing the impaled neurone by pressure ejection (10 p.s.i., 100 ms duration; Multi Channel Picospritzer, General Valve Corporation, Fairfield, NJ, USA) from a ∼50 μm tip diameter pipette. Preliminary experiments determined that 100 ms pulses were sufficient to elicit maximal membrane potential responses to each agonist. Agonists were applied at a membrane potential of −80 mV. TTX (300 nm) was present in the superfusate throughout the agonist application experiments to minimize indirect effects on membrane potential due to actions of the agonists on neighbouring perikarya and nerve terminals. However, some release of neurotransmitter from myenteric nerve terminals due to presynaptic nicotinic receptor activation has been reported to be TTX resistant (Schneider & Galligan, 2000). Also, 5-HT and ATP sometimes caused slow membrane potential depolarizations due to their agonist actions at metabotropic receptors. Nonetheless, the initial peak membrane potential response to the agonist is due to direct activation of ligand-gated ion channels (Schneider & Galligan, 2000) and that is what was measured in each neurone.
In order to examine whether the inflammation-induced participation of 5-HT in fast EPSPs was due to a change in the content of presynaptic vesicles following inflammation, we used a mouse monoclonal antibody (1: 25; Clone 5-HT-H209; Dako Cytomation, Carpinteria, CA, USA) to detect 5-HT immunoreactivity in the submucosal and myenteric plexuses (Ellis & Mawe, 2003). Segments of distal colon from control and TNBS-treated colon were placed in a Sylgard-lined dissecting dish, opened along the mesenteric border and pinned tautly with the mucosa facing up. Tissue was fixed overnight in Zamboni's fixative at 4°C. Fixative was removed by three 10 min washes in dimethylsulphoxide followed by three 10 min washes in PBS. Colon segments were dissected to yield preparations of submucosal plexus or myenteric plexus-longitudinal muscle. Prior to exposure to primary antiserum for 2 nights, all preparations were incubated for 30 min in a 10% solution of normal horse serum and 1% triton X-100 in 0.1 m sodium phosphate buffer for 30 min. Following incubation in primary antiserum, immunoreactivity was detected following incubation of preparations for 90 min in donkey anti-mouse IgG-CY3 (1: 100; Biocan Scientific, Missisauga, ON, USA). Three 10 min washes in PBS followed and finally, the preparations were mounted in glycerol buffered 0.5 m sodium carbonate buffer (pH 6.8).
Immunoreactivity was examined and documented using an Olympus Fluoview FV300 confocal microscope system on an Olympus BX50 microscope. Images of 1024 × 1024 pixels were obtained and were processed using Corel Photopaint (Corel Corporation, Ottawa, Canada). Confocal micrographs are digital composites of Z-stack scans through 1 μm optical sections with 2 × Kalman averaging.
All drugs, except for TTX (Tocris, Ellisville, MO, USA) used in the present work were obtained from Sigma-Aldrich (St Louis, MO, USA). Drugs were made up as stock solutions in distilled water and frozen in aliquots until being added directly to the superfusate on the day of the experiment. Agonists to be applied by picospritzer were dissolved in a Hepes (10 mm)-buffered saline solution (120 mm NaCl, pH 7.2) and used on the day of the experiment or frozen in aliquots for later use.