Corresponding author K. Sharkey: Department of Physiology and Biophysics, University of Calgary, 3330 Hospital Dr. NW, Calgary, Alberta T2N 4N1, Canada. Email: email@example.com
Intestinal secretion is regulated by submucosal neurones of the enteric nervous system. Inflammation of the intestines leads to aberrant secretory activity; therefore we hypothesized that the synaptic and electrical behaviours of submucosal neurones are altered during colitis. To test this hypothesis, we used intracellular microelectrode recording to compare the excitability and synaptic properties of submucosal neurones from normal and trinitrobenzene sulphonic acid (TNBS)-inflamed guinea-pig colons. Inflammation differentially affected the electrophysiological characteristics of the two functional classes of submucosal neurones. AH neurones from inflamed colons were more excitable, had shorter action potential durations and reduced afterhyperpolarizations. Stimulus-evoked fast and slow excitatory postsynaptic potentials (EPSPs) in S neurones were larger during colitis, and the incidence of spontaneous fast EPSPs was increased. In control preparations, fast EPSPs were almost completely blocked by the nicotinic receptor antagonist hexamethonium, whereas fast EPSPs in inflamed S neurones were only partially inhibited by hexamethonium. In inflamed tissues, components of the fast EPSP in S neurones were sensitive to blockade of P2X and 5-HT3 receptors while these antagonists had little effect in control preparations. Control and inflamed S neurones were equally sensitive to brief application of acetylcholine, ATP and 5-HT, suggesting that synaptic facilitation was due to a presynaptic mechanism. Immunoreactivity for 5-HT in the submucosal plexus was unchanged by inflammation; this indicates that altered synaptic transmission was not due to anatomical remodelling of submucosal nerve terminals. This is the first demonstration of alterations in synaptic pharmacology in the enteric nervous system during inflammation.
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Enteric neurones are commonly divided into two groups on the basis of functional, morphological and electrophysiological properties (Bornstein et al. 1994; Wood, 1994). One class, called S neurones, is characterized by prominent fast excitatory synaptic input, a high level of excitability and tetrodotoxin (TTX)-sensitive action potentials (APs); S neurones are the interneurones and motor neurones of enteric nerve circuits (Furness et al. 2003). The other class, called AH neurones, has TTX-resistant APs and is less excitable due to the action of a long lasting afterhyperpolarizing potential (AHP) that follows somal APs and acts to dampen excitability. AH neurones are thought to function as afferent and interneurones in enteric reflex circuits (Furness et al. 2004).
Recent studies have focused on gaining a better understanding of how interactions between the ENS and the GI immune system produce different GI tract behaviours under normal circumstances and during inflammation (De Giorgio et al. 2004; Wood, 2004). Two types of models of GI inflammation have received the most attention in studies of the ENS. One model consists of infecting guinea-pigs with GI parasites such as Trichinella spiralis, or milk-sensitizing guinea-pigs. The other model uses intraluminal administration of the hapten trinitrobenzene sulphonic acid (TNBS) to cause a cell-mediated transmural inflammation (Elson et al. 1995; Mayer & Collins, 2002). The cytokine profiles of the immune activation caused in these two models differ, but the electrophysiological effects on enteric neurones are similar in many respects: neurones are generally more excitable and synaptic transmission is altered. In the infection/sensitization model, stimulus-evoked fast excitatory postsynaptic potentials (EPSPs) are reduced upon superfusion with the sensitizing antigen (Frieling et al. 1994a,b) whereas in the TNBS model, fast EPSPs are enhanced (Linden et al. 2003).
The aim of this study was to elucidate the effects of inflammation on the electrical and synaptic properties of neurones in the colonic submucosal plexus which regulate mucosal electrolyte transport and blood flow (Vanner & Surprenant, 1991; Frieling et al. 1992; Weber et al. 2001). Inflammation-induced changes in the reflex circuitry of the submucosal plexus are likely to contribute directly to pathophysiological alterations in mucosal secretion and absorption, as well as the integrity of the epithelial barrier (Neunlist et al. 2003). This is the first investigation of the effects of cell-mediated inflammation on the electrical and synaptic properties of submucosal neurones. Data from this investigation indicate that AH neurones are hyperexcitable in the submucosal plexus of the inflamed colon, and that synaptic facilitation occurs through a mechanism that involves recruitment of additional neurotransmitters.
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.
Statistical analyses were carried out using Prism 3 (Graphpad Software, San Diego, CA, USA). Differences between neurones from normal and inflamed animals were detected using unpaired two-tailed Student's t tests. Non-parametric values were compared using Fisher's exact test or the Mann-Whitney test. A P value of < 0.05 was considered statistically significant. Summary data were plotted using Prism 3.
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.
Over the course of 6 days, TNBS-treated animals gained significantly less weight than controls (20.9 ± 4.4 g (n= 32; mean ±s.e.m.) versus 42.5 ± 4.0 (n= 36); P= 0.004, unpaired Student's t test). In addition to gaining less weight than controls, TNBS-treated animals had significantly higher macroscopic damage scores than controls (6.5 ± 0.7 versus 0.75 ± 0.06, n= 32 and 36, respectively; P= 0.0008, Mann-Whitney test). Electrophysiological characteristics of S and AH neurones that were not affected by inflammation are detailed in Tables 1 and 2. In general, the excitability of S neurones was unchanged and the properties of slow synaptic potentials recorded in AH neurones were unaffected by inflammation.
Table 1. Electrophysiological characteristics of AH neurones that were not altered by inflammation
Mean ±s.e.m. data are shown. Number of neurones for each measurement is given in parentheses.
−60 ± 0.9 (14)
−57.7 ± 1.4 (15)
Input resistance (MΩ)
193.4 ± 30.7 (14)
196 ± 22.2 (15)
Incidence of anode break APs (%)
Incidence of spontaneous APs (%)
Incidence of fast EPSPs (%)
AP threshold (pA)
142 ± 26.2 (14)
98 ± 13.8 (15)
APs at rheobase
1.07 ± 0.07 (14)
1.26 ± 0.16 (15)
AP amplitude (mV)
80.3 ± 1.9 (14)
74.4 ± 2.9 (15)
Fast EPSP incidence (%)
Fast EPSP amplitude at RMP (mV)
13.5 ± 2.1 (4)
Slow EPSP incidence (%)
Slow EPSP amplitude
9.75 ± 2.0 (9)
7.19 ± 1.9 (9)
Table 2. Electrophysiological characteristics of S neurones that were not altered by inflammation
Mean ±s.e.m. data are shown. Number of neurones for each measurement is given in parentheses.
−49.4 ± 0.9 (38)
−48.3 ± 0.8 (36)
Input resistance (MΩ)
211 ± 13.9 (38)
239 ± 21 (36)
AP threshold (pA)
90.6 ± 9.6 (38)
76.7 ± 8.4 (36)
APs at rheobase
1.63 ± 0.14 (38)
1.92 ± 0.26 (36)
APs at 2 × rheobase
8.48 ± 1.05 (38)
8.78 ± 1.0 (36)
16.97 ± 1.73 (38)
20.4 ± 1.72 (36)
AP amplitude (mV)
61.0 ± 1.8 (38)
65 ± 1.5 (36)
1.44 ± 0.06 (38)
1.47 ± 0.06 (36)
Slow EPSP incidence (%)
Slow IPSP incidence (%)
Slow IPSP amplitude
13.7 ± 1.4 (8)
17.8 ± 2.9 (7)
Anode break APs (%)
Spontaneous APs (%)
The morphologies of neurones that were evaluated electrophysiologically and sufficiently filled with biocytin to determine cell shape and axonal projections were analysed. Nine of fourteen AH neurones from control preparations and 10 of 15 AH neurones from inflamed submucosal plexus were identified following fixation and processing to reveal biocytin. These neurones had smooth round cell bodies with several axonal processes (Lomax et al. 2001). In many cases, axons of AH neurones could be followed for distances of three to eight ganglia where they gave rise to varicose branches that provided dense innervation of the ganglia in question. Twenty three of thirty eight control S neurones and 21 of 36 inflamed S neurones were located. All S neurones were uniaxonal and displayed a variety of somal morphologies, as previously described (Lomax et al. 2001). The axons of S neurones could only be traced for short distances before the axons left the plane of the submucosal plexus, presumably to innervate the secretory epithelium. Due to the short distances that axons of submucosal S neurones could be traced, we were unable to characterize S neurones as orally, aborally or circumferentially projecting. We also were unable to characterize submucosal neurones according to their neurochemical phenotype as we concluded from preliminary immunohistochemical experiments that immunoreactivity for nitric oxide synthase (NOS), calretinin, neuropeptide Y (NPY) and calbindin was much reduced in the submucosal plexus following in vitro experiments compared with submucosal preparations that were fixed immediately.
The resting membrane potential and input resistance of AH neurones were unchanged following TNBS colitis (Table 1). There was also no change in the amplitude of slow EPSPs recorded in AH cells from inflamed colon. However, the incidence of fast EPSPs recorded in response to single presynaptic stimuli increased from 1 of 14 neurones in control preparations to 4 of 15 neurones following inflammation. This increase did not reach statistical significance (Fisher's exact test). Two AH neurones from inflamed colons that received fast synaptic input were superfused with 100 μm hexamethonium, and in both cases the fast EPSP was substantially reduced but not completely abolished.
The most marked difference between AH neurones from control and inflamed colons was that AH neurones from inflamed colons were more excitable. This was evident as an increase in the number of APs at 2 × rheobase and in the maximum number of APs each neurone was capable of firing during a 500 ms depolarization (Fig. 1A). Associated with this increase in excitability during inflammation was a decrease in the amplitude (3.2 ± 0.5 versus 6.0 ± 0.6 mV; P= 0.0076) and magnitude (Fig. 1B) of the AHP in neurones from inflamed colon.
Action potential durations at half-amplitude (APD50) were measured from intracellularly stimulated APs (2 ms duration, 200–500 pA current amplitude) and from antidromically activated APs. AP parameters using both stimuli were compared in 12 AH neurones, 6 each from control and inflamed submucosal plexuses. AP amplitudes from intracellularly and antidromically activated APs were 73.2 ± 2.0 and 72.6 ± 1.9 mV, respectively (P= 0.57; paired t test). APD50s from intracellularly and antidromically activated APs were 1.60 ± 0.12 and 1.57 ± 0.09 ms, respectively (P= 0.29, paired t test). Because there were no differences between measurements taken from antidromically and intracellularly stimulated APs in AH neurones, comparisons between normal and inflamed colon were made with APs generated by brief intracellular current pulses. This was because all AH neurones fired APs in response to intracellular stimulation but not every AH neurone fired antidromic APs in response to internodal strand stimulation. The APD50 was significantly shorter in inflamed animals than in controls (Fig. 1C and D). Reduced AP duration may result in less Ca2+ influx during the AP and this could lead to less activation of the calcium-activated potassium conductance (gKCa) that underlies the AHP. There was a significant correlation between APD50 and AHP magnitude in AH neurones from control and inflamed colons (Fig. 2A). A significant correlation also existed between the maximum number of APs an AH neurone could fire and its APD50 (Fig. 2B).
We also examined the possibility that an increase in a hyperpolarization-activated non-selective cation conductance (IH) might contribute to the decrease in AHP observed in the present study. Activation of IH causes a ‘sag’ in the electrotonic potential in response to hyperpolarizing current injection. At a given input resistance, the amplitude of the sag gives an estimate of the amplitude of the IH current. We measured the sag that occurred during 500 ms hyperpolarizations applied in 20 pA increments in control and inflamed AH neurones. We found no significant difference in the amplitude of the sag (n= 14 control, 15 inflamed AH neurones), and did not detect any difference in the component of hyperpolarizations unmasked by superfusion with 2 mm CsCl between control and inflamed AH neurones (n= 4 neurones each).
As described above, no changes in the passive or active electrical properties of S neurones were detected in the inflamed colon. However, the amplitudes of stimulus-evoked fast and slow EPSPs recorded in S neurones were significantly increased during inflammation (Fig. 3A and B). Spontaneous fast EPSPs were significantly (P= 0.002; Fisher's exact test) more common in S neurones from inflamed colons compared with controls (Fig. 3C and D). We examined the sensitivity of spontaneous fast EPSPs to nicotinic receptor blockade with 100 μm hexamethonium. In two S neurones from control colon and three S neurones from inflamed colon, hexamethonium blocked spontaneous fast EPSPs. This indicates that the increase in spontaneous fast EPSPs during inflammation was due to enhanced acetylcholine release. Spontaneous APs that were not due to fast synaptic input were observed in 4 of 38 S neurones from control colons and in 4 of 36 S neurones from inflamed colons. Spontaneous AP generation in four of these neurones (2 each from control and inflamed colons) was quenched by hyperpolarizing the membrane potential, which suggests that the site of generation of these APs was located close to or on the cell body of these neurones (Spencer & Smith, 2004). We also observed spontaneous slow depolarizations that resembled electrically evoked slow EPSPs in four S neurones from inflamed colons; spontaneous slow depolarizations were not detected in control colons. In addition, previous reports of the electrophysiological properties of submucosal neurones from normal guinea-pig distal colon never documented the occurrence of these slow depolarizations (Frieling et al. 1991; Lomax et al. 2001). In two neurones, spontaneous slow depolarizations were preceded by a burst of spontaneous fast EPSPs, indicating spontaneous release of excitatory transmitter immediately before the onset of the slow depolarization (Fig. 3E).
Fast EPSPs in control neurones were almost entirely sensitive to the nicotinic receptor antagonist hexamethonium (100 μm) whereas in inflamed neurones a substantial hexamethonium-resistant component persisted (Fig. 4). In control preparations, 4 of 14 (28.6%) fast EPSPs recorded in S neurones had a hexamethonium-resistant component, as compared with 14 of 18 (77.7%; P= 0.011, Fisher's exact test) in S neurones from inflamed colons. No difference was detected in the amplitude of the hexamethonium-sensitive component of fast EPSPs in these preparations (Fig. 4C), indicating that the amplitude of the cholinergic component of fast EPSPs was not altered in the inflamed colon. We then utilized antagonists of 5-HT3 and P2X receptors, which contribute to fast EPSPs at myenteric synapses (Galligan, 2002). For these studies, granisetron (1 μm) and PPADs (30 μm) were used to block 5-HT3 receptors and P2X receptors, respectively. In control preparations, these compounds had no significant effect on the amplitude of fast EPSPs recorded at −80 mV (19.8 ± 2.3 mV before versus 19.0 ± 2.2 mV after PPADs, P= 0.78; 20.6 mV ± 3.0 mV before versus 20.1 ± 3.3 mV after granisetron, P= 0.62, paired Student's t tests). However, in the inflamed colon, both antagonists significantly reduced the amplitude of fast EPSPs (27.5 ± 2.1 mV before versus 22.4 ± 2.6 mV after PPADs, P= 0.001; 35.83 ± 2.5 mV before versus 29.2 ± 2.8 mV after granisetron, P= 0.015). In addition, the amplitudes of granisetron- and PPADs-sensitive components of the fast EPSP, measured as the difference between the amplitude of fast EPSPs in control conditions versus the amplitude during superfusion with the antagonist, were significantly higher in inflamed colons than control colons (Fig. 4C).
In order to determine whether the increased participation of 5-HT3 receptors and P2X receptors in fast EPSPs during inflammation involved an increase in the postsynaptic sensitivity to 5-HT and ATP, we evaluated the effects of exogenously applied agonists on the membrane potential of S neurones in control and inflamed preparations. We found no significant differences in the membrane potential response of neurones from control or inflamed colons to ACh, ATP or 5-HT (Fig. 5).
We also examined the paired pulse ratio (PPR) of fast EPSPs separated by a 50 ms interval to determine whether there might be differences in the ability of fast excitatory synapses to undergo short-term changes in synaptic efficacy. Also, if the release of transmitter increased during inflammation to the extent that it exhausted the readily releasable pool of neurotransmitter in presynaptic terminals, this might be manifest as a decrease in PPR during inflammation. There was a small but statistically insignificant increase in the PPR in S neurones following inflammation (0.88 ± 0.12 versus 1.01 ± 0.07, respectively, n= 7 control and 9 inflamed; P= 0.32, unpaired t test).
We next used immunohistochemical techniques to localize 5-HT in inflamed and control myenteric and submucosal tissue to examine whether we could detect any changes in 5-HT immunoreactivity during inflammation. In the myenteric and submucosal plexuses of control preparations, 5-HT immunoreactivity was observed in smooth fibres that coursed through ganglia and in varicose axons that appeared to surround neurones within ganglia. In the myenteric plexus, faint immunoreactivity was observed in nerve cell bodies (data not shown; see Lomax & Furness, 2000). As there are no 5-HT immunoreactive cell bodies in the submucosal plexus of the guinea-pig colon, it seems likely that the 5-HT immunoreactive cell bodies in the myenteric plexus are the source of 5-HT immunoreactive nerve terminals in the submucosal plexus (Lomax & Furness, 2000). We did not observe any marked alterations in the intensity or distribution of 5-HT immunoreactivity in the submucosal plexus following inflammation (Fig. 6; n= 3 control and 3 TNBS colitis animals).
The aim of this study was to examine the effects of cell-mediated inflammation on the electrical and synaptic properties of submucosal neurones. We utilized the TNBS model of colitis in guinea-pigs, which is thought to lead to similar immune activation profiles to those seen in Crohn's disease (Strober et al. 2002). Our data indicate that dramatic changes occur in the electrical and synaptic properties of submucosal neurones of the inflamed colon. The changes that we observe in synaptic transmission appear to be restricted to S neurones whereas inflammation-induced changes in neuronal excitability are restricted to AH neurones. This suggests that gastrointestinal inflammation can selectively alter the cell body properties of one class of neurones and the synaptic properties of another class.
In previous studies of the effects of inflammation or infection on enteric neurophysiology, the most consistent finding was enhanced excitability of AH neurones (Frieling et al. 1994a,b; Palmer et al. 1998; Linden et al. 2003; Liu et al. 2003). This was evident as an increase in the number of APs fired during a depolarization and was often accompanied by an increase in the number of neurones displaying anode break excitation. AH neurones in the submucosal plexus are thought to be primary afferent neurones in enteric nerve circuits and as such are the initiators of a variety of reflex responses to stimuli (Pan & Gershon, 2000; Furness et al. 2004). An increase in their excitability may have profound consequences for the function of these reflex circuits, leading perhaps to excessive and inappropriate reflex responses or even desensitization of downstream synapses.
Results of a previous study of neuroplasticity associated with TNBS-induced colitis indicated that enhanced myenteric AH neurone excitability was associated with a decrease in the magnitude of the AHP (Linden et al. 2003). We saw similar changes in submucosal neurones. The increase in myenteric AH neurone excitability during TNBS colitis appears to be due to products of cyclooxygenase-2 (COX-2), as treating guinea-pigs with a COX-2 antagonist blocks the increase in AH neurone excitability during colitis (Linden et al. 2004), and prolonged exposure to prostaglandin E2 leads to an increase in AH neurone excitability (Manning et al. 2002).
The AHP in AH neurones is due to the activation of a calcium-activated potassium conductance (Hirst et al. 1985; North & Tokimasa, 1987; Vogalis et al. 2001, 2002). Upon activation, the gKCa hyperpolarizes the membrane potential and dampens excitability. Membrane potential hyperpolarization leads to the activation of a hyperpolarization-activated cation conductance (IH), which antagonizes the inhibitory effect of the gKCa (Galligan et al. 1990). Linden et al. (2003) provided evidence that the decrease in the AHP could be due to increased IH. In the present study, we observed an association between AH neurone excitability and AHP magnitude. However, in the present study, the APD50 of AH neurones was reduced during inflammation. The duration of action potentials is a major determinant of AHP amplitude; shorter action potentials lead to less influx of Ca2+ via voltage-gated Ca2+ channels, which reduces calcium-induced calcium release from ryanodine receptors, resulting in reduced activation of gKCa (Hillsley et al. 2000). Thus we consider that the mechanism of enhanced excitability in submucosal AH neurones during inflammation involves a decrease in APD50. Previous electrophysiological studies have shown that increasing the APD in myenteric AH neurones from the guinea-pig small intestine increases the AHP (Hirst et al. 1985; North & Tokimasa, 1987). Consistent with this concept, significant correlations existed between APD and AHP magnitude, and between APD and excitability (Fig. 2). Data from a combined calcium-imaging and electrophysiology study in AH neurones of the guinea-pig ileum support this notion. A significant correlation between the peak intracellular calcium concentration and the amplitude of the AHP was reported (Hillsley et al. 2000).
In this study we detected a significant increase in the amplitude of evoked fast EPSPs in submucosal S neurones from TNBS-inflamed colons. During infection with Trichinella spiralis, and following sensitization with bovine milk, the amplitude of fast EPSPs was reduced in enteric neurones of the guinea-pig colon when preparations were superfused with the sensitizing antigen (Frieling et al. 1994a,b). In a previous study (Linden et al. 2003), evoked fast EPSPs in myenteric S neurones increased in amplitude following TNBS colitis; however, it was not determined whether this increase was associated with a change in the pharmacology of the synaptic potentials. The results of the present study indicate that the increase in fast EPSP amplitude in the submucosal plexus following inflammation is due to a change in the neurotransmitters that contribute to fast EPSPs.
The fast EPSPs recorded following extracellular stimulation of interganglionic connectives probably are compound fast EPSPs due to release of transmitters from a number of nerve terminals, including terminals of submucosal primary afferent neurones and myenteric primary afferent and interneurones (Furness et al. 2003). In the normal guinea-pig colon myenteric plexus, the pharmacology of compound fast EPSPs is complex, with ACh, ATP and 5-HT thought to participate (Galligan, 2002; Nurgali et al. 2003). By contrast, in the normal guinea-pig colon submucosal plexus the majority of evoked fast EPSPs are purely cholinergic (Frieling et al. 1991; Lomax et al. 2001). The relatively simple pharmacology of fast EPSPs in the submucosal plexus compared with the myenteric plexus has enabled the identification of the pharmacological differences found in the present work.
Although there was a difference in the pharmacology of fast EPSPs, it does not appear to involve changes in the expression of ionotropic receptors on postsynaptic membranes following colitis. This conclusion is based on the finding that depolarizations in response to exogenously applied ACh, ATP and 5-HT were not different between S neurones from normal and inflamed colons. Interestingly, many S neurones from control and inflamed colon were sensitive to all three agonists, despite the fact that 5-HT and ATP typically did not contribute to fast EPSPs under control conditions. It is possible that the agonists applied by pressure microejection could access and activate synaptic plus extrasynaptic receptors, and that while the absolute number of receptors is unaltered by inflammation, the proportion of those receptors that are trafficked to synaptic sites apposing presynaptic varicosities is increased by inflammation.
If facilitation of fast synaptic transmission involves presynaptic modulation a number of potential mechanisms could contribute. It is possible that inflammation changes the innervation patterns of axons that contain ATP and 5-HT such that axons containing these neurotransmitters now innervate submucosal S neurones whereas in control conditions this is not the case. There are no reliable markers of purinergic nerve terminals, but the finding that 5-HT immunoreactivity is not altered by colitis (Fig. 6) would argue against this possibility. Up-regulation of the release of neurotransmitters from nerve terminals that release ATP and 5-HT following inflammation may also explain our findings. Another potential mechanism of the observed changes is an increase in the calcium sensitivity of the release mechanism. This increase in calcium sensitivity may lead to vesicles containing 5-HT and ATP being released during inflammation whereas in control neurones, only ACh is released in response to single presynaptic APs. A selective reduction in potassium conductance in nerve terminals during inflammation might also contribute to the observed effects on synaptic transmission. A decrease in potassium conductance, which would increase the APD in the nerve terminal, would result in more calcium entering the nerve terminal and lead to increased neurotransmitter release. Preliminary data on myenteric fast EPSPs during TNBS colitis identified a role for a decrease in iberiotoxin-sensitive gKCa located on nerve terminals in the fast EPSP amplitude (D. R. Linden, K. A. Sharkey and G. Mawe, personal communication).
Effects of inflammation on synaptic transmission have been reported in the pain pathways of the dorsal horn of the spinal cord and the rostral ventral medulla (Robinson et al. 2002; Furue et al. 2004). The effects on synaptic transmission observed in these central systems were in response to inflammation distant from the affected synapses, whereas in the present work, TNBS colitis results in transmural inflammation. This may lead to exposure of enteric synapses to an array of inflammatory mediators such as prostaglandins, interleukins and reactive oxygen species. It is interesting to note that in the myenteric plexus, inhibition of COX-2 in TNBS-treated animals restored excitability and the AHP in AH neurones to their normal levels, but inflammation-induced synaptic facilitation persisted (Linden et al. 2004). These data suggest that inflammation alters cellular excitability and synaptic transmission by distinct mechanisms. One potential mechanism for the increase in synaptic transmission in the ENS during colitis is an increase in the in vivo frequency of action potential discharge. In the dorsal horn of the spinal cord, it has been shown that increased AP frequency, due to noxious stimuli or excessive colonic distension, can lead to an increased amplitude of synaptic currents due to a phenomenon known as synaptic windup (Mayer & Gebhart, 1994). It is conceivable that a similar paradigm may exist within enteric reflex circuits.
In conclusion, TNBS-colitis leads to specific changes in excitability and synaptic transmission that are restricted to particular classes of submucosal neurones. Afferent neurones become more excitable while motor neurones receive larger synaptic potentials and are more often in receipt of spontaneous fast EPSPs. Inflammation-induced changes to the signalling pathways in the submucosal plexus may lead to altered release of secretagogue from the nerve terminals of submucosal secretomotor/vasomotor neurones, which might lead to changes in ion transport and blood flow, leading to the symptoms observed in inflammatory bowel disease. Chronic up-regulation of enteric signalling pathways might lead to receptor desensitization, which could also result in functional changes in the GI tract in states of inflammation. Identification of the mechanisms whereby inflammation leads to such profound changes in components of enteric reflex circuits may lead to treatments for some of the symptoms of inflammatory bowel diseases.
This work was supported by an operating grant from the Crohn's and Colitis Foundation of Canada (K.S. and G.M.), and by NIH grant DK62267 (G.M.). K.S. is an Alberta Heritage Foundation for Medical Research Medical Scientist and A.L. is the recipient of a Canadian Association of Gastroenterology/Astra Zeneca/Canadian Institutes of Health Research postdoctoral fellowship.