Abstract
Changes in primary sensory neurons are likely to contribute to the emergence of chronic visceral pain. An important step in understanding visceral pain is the development of comprehensive phenotypes that combines functional and anatomical properties for these neurons. We developed a novel ex vivo physiology preparation in mice that allows intracellular recording from colon sensory neurons during colon distension, in the presence and absence of pharmacologic agents. This preparation also allows recovery of functionally characterized afferents for histochemical analysis. Recordings obtained from L6 dorsal root ganglion cells in C57BL/6 mice identified two distinct populations of distension-responsive colon afferents: high-firing frequency (HF) and low-firing frequency (LF) cells. Fluid distension of the colon elicited rapid firing (>20 Hz) in HF cells, whereas LF cells seldom fired >5 Hz. Distension response thresholds were significantly lower in HF cells (LF, 17.5 ± 1.1 cmH2O; HF, 2.6 ± 1.0 cmH2O). Responses of most LF afferents to colon distension were sensitized by luminal application of capsaicin (1 μm; 8 of 9 LF cells), mustard oil (100 μm; 10 of 12 LF cells), and low pH (pH 4.0; 5 of 6 LF cells). In contrast, few HF afferents were sensitized by capsaicin (3 of 9), mustard oil (2 of 7), or low pH (1 of 6) application. Few HF afferents (4 of 23) expressed the capsaicin receptor, TRPV1. In contrast, 87% (25 of 29) of LF afferents expressed TRPV1. TRPV1 has been shown to be required for development of inflammatory hyperalgesia. These results suggest a unique functional role of TRPV1-positive colon afferents that could be exploited to design specific therapies for visceral hypersensitivity.
Introduction
Acute abdominal discomfort and pain is often triggered by inflammation or other identifiable injury. However, most patients with chronic abdominal pain do not show clear structural or biochemical abnormality, leading to a diagnosis of irritable bowel syndrome, dyspepsia, or other functional disease. Changes in visceral sensation such as hypersensitivity and hypervigilance likely contribute to common functional disorders, and visceral sensory input is a potentially important treatment target. Spinal afferents innervating the proximal or distal gastrointestinal tract have been characterized by response to organ distension in vivo as low-threshold and high-threshold afferents (Sengupta and Gebhart, 1994a; Ozaki and Gebhart, 2001). Sixty to 70% of spinal afferents are low threshold and respond to distension pressures <10 mmHg. High threshold afferents, in contrast, respond to distension pressures >30 mmHg; these higher pressures correspond with the onset of nocifensive responses, suggesting a role for high threshold afferents in nociception (Ness and Gebhart, 1988; Naliboff et al., 1997; Ozaki et al., 2002). Gastrointestinal afferents have been characterized in vitro as mucosal, muscular, or serosal by distinct mechanical response profiles, although these designations have not yet been confirmed by sufficiently detailed anatomical studies (Lynn and Blackshaw, 1999; Brierley et al., 2004). Despite divergent responses to mechanical stimuli, the majority of visceral sensory afferents is polymodal and also responds to thermal and chemical stimuli.
Mechanical sensitivity of visceral afferents is altered in mice lacking functional transient receptor potential vanilloid receptor 1 (TRPV1) channels (Birder et al., 2002; Rong et al., 2004; Jones et al., 2005; Daly et al., 2007). These results predict a role for TRPV1, a nonselective cation channel responsive to noxious heat, low pH, and vanilloid compounds such as capsaicin (CAP) and anandamide (Caterina et al., 1997, 2000; Tominaga et al., 1998; Akopian et al., 2008; Ruparel et al., 2008), in visceral sensation. TRPV1 channels may detect stretch directly, via mechanical deformation of sensory neuronal endings or adjacent tissues, or indirectly, via compounds such as ATP released from surrounding tissues during stretch (Cooke et al., 2004; Koizumi et al., 2004). Alternatively, TRPV1 may modulate the overall sensitivity of polymodal visceral nociceptors to multiple stimuli.
In the present study, we describe a novel ex vivo preparation for intracellular recording of intact visceral afferents. Functionally characterized neurons were classified immunohistochemically, after intracellular neurobiotin injection via the recording electrode. We demonstrate that mechanically sensitive mouse colon afferents segregate into two groups based on firing frequency and distension response threshold. High firing-frequency neurons (HF) fire on approximately four times faster than low firing-frequency afferents (LF), exhibit greater adaptation, and have significantly lower distension response thresholds (p < 0.001; HF, 2 ± 1 cmH2O; LF, 17 ± 1 cmH2O). Surprisingly, the majority (87%) of HF afferents were TRPV1-negative, whereas nearly all (87%) LF afferents expressed TRPV1 and could be sensitized by capsaicin, mustard oil (MO), and/or acid. These data suggest that TRPV1 channel activity modulates a specific subset of visceral afferents.
Materials and Methods
Animals.
All experiments were performed in male and female mice (34 males; 16 females), 3–6 weeks of age, housed in a 12 h light/dark cycle with ad libitum access to water and food at the certified animal care facility of the University of Pittsburgh. All mice were bred in our vivarium. Animal handling adhered to the Guide for the Care and Use of Laboratory Animals (National Research Council); all procedures were approved by the Institutional Animal Care and Use Committee of the University of Pittsburgh. Two strains of wild-type mice were used: 30 C3H/C57BL/6 hybrids (maintained at the University of Pittsburgh) and 20 C57BL/6 (originally from The Jackson Laboratory but bred for several generations in our facility). There was no difference in the data collected between the two lines of mice, and therefore data were pooled from all experiments.
Ex vivo preparation.
Mice were killed using an overdose of isoflurane, injected with heparin, and transcardially perfused with 30 ml of carboxygenated, ice-cold (∼9°C) artificial CSF (sucrose-ACSF). To prevent excitotoxicity, sodium chloride was replaced by an iso-osmolar concentration of sucrose (sucrose-ACSF) (in mm: 253.9 sucrose, 1.9 KCl, 1.2 KH2PO4, 1.3 MgSO4, 2.4 CaCl2, 26.0 NaHCO3, 10.0 d-glucose). A laminectomy was then performed to expose lumbar and sacral spinal cord and dorsal root ganglia. The lower one-half of the animal was then removed to a separate dissection chamber circulating cold sucrose-ACSF. All abdominal organs except the colon were then removed. The colon was detached from the small intestines ∼1 cm distal to the cecum. Polyethylene tubing (PE 50) attached to a 10 cc syringe containing perfusion solution was inserted into the anus and pressure was applied to expel fecal pellets through the proximal end of the colon. The pubic bone was split at the pubic symphysis, and the colon was freed from ventral attachments. The central primary ramus of one of the L6 spinal nerves (typically the left) was dissected free of the surrounding tissue and traced into the pelvic nerve. The colon was dissected free of all dorsal and lateral attachments except for the pelvic nerve and connective tissue surrounding the nerve. The vertebral column was reduced to bone, connecting ligaments, and the spinal cord adjacent to the L3–S1 spinal ganglia. The entire preparation was then moved into the recording dish perfused with chilled (∼9°C) and carboxygenated ACSF (in mm: 127 NaCl, 1.9 KCl, 1.2 KH2PO4, 1.3 MgSO4, 2.4 CaCl2, 26.0 NaHCO3, 10.0 d-glucose). The temperature was slowly raised to 33°C. The rectum was intubated with PE tubing connected to a gravity-driven perfusion system for distension to 10, 20, 30, and 40 cmH2O (see Fig. 1). PE tubing was also inserted into the proximal end of the colon for drainage. This tubing was connected to a pressure transducer to monitor intracolonic pressure during distension. The position of proximal and distal tubes was secured through silk ligatures. The ventral rami of the L6 spinal nerve was traced to its entry point into the pelvic nerve, where a suction electrode was placed for electrical stimulation ∼3 mm distal to L6 DRG. Typically, the time from beginning perfusion to initiating recordings was ∼2.5 h.
Electrophysiologic recordings.
A total of 50 mice was used for these studies (30 C3H/C57BL/6 and 20 C57BL/6). Both male (34) and female (16) mice were analyzed. There was no strain or sex effect, and therefore all data were pooled. Recordings were made from individual L6 neurons using sharp borosilcate glass electrodes (R > 100 MΩ) filled with 1.0 m potassium acetate and 5% Neurobiotin (Vector Laboratories). Intracellular penetrations were recognized by a sudden voltage change. Electrophysiological responses were acquired with an Axoclamp 2B amplifier, sampled at 25 kHz, digitized (CED Micro 1401; Cambridge Electronic Design) and stored on a computer (Gateway) using Spike 2 software (Cambridge Electronic Design). Cells generating an action potential in response to electrical stimulation of the pelvic nerve were tested with brief colon distension (<30 cmH20 for 5 s) and probing with a glass rod. If responses were elicited by either stimulus, a stepwise colon distension series was performed to pressures of 10, 20, 30, and 40 cmH2O with oxygenated bath solution. Each distension pressure lasted 20 s, followed by drainage of the colon and a 20 s rest period before the next distension. In initial experiments, the distension series was repeated two to three times to determine whether colon afferents exhibited sensitization or habituation in response to repeated colon distension. Neither was seen in any of the cells examined in this manner (data not shown). Once the distension series was completed, the colon was drained and probed with von Frey hairs to identify receptive fields. After the initial distension series and von Frey probing, the cell was filled intracellularly with Neurobiotin (current injection; 1 nA, 60 s) for subsequent immunohistochemical identification. An additional distension series was then performed to examine the effect of Neurobiotin labeling and current injection on cellular response properties (action potential properties including threshold, distension response threshold, stimulus response function). Cells were rarely seen in which labeling altered response properties; these cells were excluded from additional experiments.
The colon was then infused with one or more of the following solutions: ACSF, pH 4, 5% DMSO (vehicle), 1 μm capsaicin in 5% DMSO, or 100 μm mustard oil in 5% DMSO. For each infusion, 3 ml of solution was infused into the colon over 1–2 min. Solutions were drained from the proximal end of the colon at the same rate that they were introduced into the rectum to minimize colon distension during drug infusion. A colon distension series was then performed after infusion of each test solution. The 5% DMSO control solution was tested before solutions containing capsaicin or mustard oil were infused, to examine vehicle effects on each afferent. At the end of the 10 min testing period, the colon was washed with 10 ml of oxygenated ACSF, and a distension series was run again to ensure that the cell had returned to baseline predrug responses. If effects could not be washed out, the experiment was terminated. In some cases, cells penetration was maintained for over an hour, allowing testing of a single cell with multiple solutions. Presentation order of capsaicin, mustard oil, and acid was varied between cells in which multiple sensitizing agents were delivered. Washout was obtained between each drug, and no significant differences correlated with presentation order were seen.
Application of drugs might produce alterations in colon compliance; changes in mechanical properties of the colon could modulate neuronal response properties. To test this possibility, the amount of fluid needed to maintain constant pressure within the colon was measured at each distension pressure for each drug infusion. We tested colon compliance by serially individually infusing each of the drugs used in this study in the order that was used for physiological recording experiments: ACSF, 5% DMSO, 1 μm capsaicin (in 5% DMSO), 100 μm mustard oil (in 5% DMSO), ACSF (to wash out drugs and DMSO), and ACSF, pH 4. Distension series were run four times in the presence of each drug, and the results were averaged.
Data analysis.
For each cell, the resting membrane potential, conduction velocity, spike amplitude, action potential duration at 50% of peak amplitude, and amplitude and duration of the afterhyperpolarization was determined as described previously (Bielefeldt et al., 2006). To generate stimulus response functions, both the mean action potential frequency for the entire stimulation period and maximal instantaneous frequency were determined for each distension pressure.
Immunohistological analysis of recorded cells.
At the end of the experiment, the L6 DRG was removed and immersion fixed [4% paraformaldehyde in 0.1 m PB (in mm: 12.5 NaH2PO4, 87.5 Na2HPO4) for 20 min at 25°C]. Ganglia were then embedded in 10% gelatin, postfixed in 4% paraformaldehyde for 30 min, followed by cryoprotection in 20% sucrose for at least 1 h. Frozen sections (35 μm) were collected serially in PB and reacted with CY2-tagged avidin to label Neurobiotin-filled cells (Vector). Each section was stained with rabbit anti-TRPV1 (Neuromics), and goat anti-GFRα3 (R&D Systems). After incubation in primary antiserum, tissue was washed and incubated in donkey anti-rabbit and donkey anti-goat secondary antiserum (conjugated to CY3 or CY5, respectively, diluted 1:500; Molecular Probes). Distribution of fluorescent staining was determined using an Olympus confocal microscope and sequential scanning to prevent bleedthrough of the different fluorophores. Specific staining for both antibodies was tested on DRG tissue from appropriate TRPV1−/− or GFRα3−/− mice. No staining for either antibody was observed in knock-out tissue (Fasanella et al., 2008).
Statistical analysis.
All physiology data are mean ± SE. Results were analyzed using the Mann–Whitney rank sum test, a two-way ANOVA, or Fisher's exact test where appropriate. A value of p < 0.05 was considered statistically significant.
Results
Basic properties of colon afferents
For these studies, we used a novel ex vivo electrophysiology preparation in which recordings were obtained from sensory neuron cell bodies in the L6 spinal ganglion in response to stimulation of the colon with fluid distension or von Frey probing. The preparation is described in Figure 1. A total of 127 colon afferents were identified by response to colon distension and/or probing of the colon with von Frey hairs. In addition, action potentials were recorded from 221 afferents electrically stimulated by the pelvic nerve suction electrode, but that were nonresponsive to colon stimulation (nonresponsive afferents). All colon afferents conducted in the C-fiber range (conduction velocity, ≤0.41 m/s); no difference in conduction velocity was observed between colon and nonresponsive pelvic afferents (Table 1). Most colorectal afferents were quiescent at rest; only 20% (27 of 127) of colon sensory neurons were spontaneously active (mean spontaneous firing rate, 1.5 ± 0.1 Hz). Sixty-six percent (51 of 77) of colon afferents responded to von Frey probing; the average threshold for response was 4.1 ± 0.1 g. All colon afferents responsive to von Frey probing also responded to colon distension.
Action potential shape was not significantly different between all colon afferents and nonresponsive pelvic afferents. However, classification of colon afferents by response to distension as HF and LF afferents (see below) revealed significant differences in action potential shape. Action potentials recorded from HF afferents were of significantly (p < 0.05) briefer duration than those recorded from LF afferents [action potential duration at half-amplitude (APD50): HF, 1.81 ± 0.1 ms; LF, 2.3 ± 0.1 ms]. This difference, illustrated in Figure 2, may underlie the higher firing rates seen in HF cells (Hille, 2001). Previous studies in cutaneous afferents have demonstrated a link between somal action potential shape and sensory receptive field properties, although the precise channel mechanism is unknown (Fang et al., 2005, 2006).
Responses to colorectal distension
All 127 colon afferents encoded stimulus intensity over the entire suprathreshold range of colon distension tested. Based on the stimulus response profile to colon distensions from 10 to 40 cmH2O, two distinct populations of afferents could be distinguished. The first group contained afferents that responded to colon distension with high-frequency action potential firing. These were characterized as HF cells. Responses obtained from a representative HF colon afferent are shown in Figure 2. The Neurobiotin-labeled cell body of the HF afferent (Fig. 2A, green) was identified as negative for the ion channel TRPV1 (Fig. 2A, red) and the growth factor receptor GFRα3 (Fig. 2A, blue) expression by post hoc immunocytochemistry. In HF cells, maximal colon distensions (40 cmH2O) elicited action potential firing at rates >20 Hz (Fig. 2B). Responses in ∼50% of HF cells showed significant adaptation of firing rate during colon distension, such that the firing rate at end of 20 s of colon distension (terminal firing rate) was <50% of the peak firing rate. Firing rate adaptation in HF cells was 9.5 ± 1.1 Hz or 48 ± 5% (terminal firing rate/peak firing rate).
The second group of colon afferents responded to colonic distension with low-frequency action potential firing. These were characterized as LF cells, and responses obtained from a representative LF colon afferent are shown in Figure 2. Post hoc immunohistochemical examination of this LF afferent (Fig. 2B, green) identified this cell as TRPV1 (Fig. 2B, red) and GFRα3 (Fig. 2B, blue) expressing. Although maximal colonic distension (40 cmH2O) elicited robust action potential firing in LF cells, action potential firing rates were much slower (<10 Hz) than those recorded in HF cells in response to the same distension stimulus (Fig. 2, compare A, B). Responses in 70% of LF cells showed little or no adaptation of firing rate during the 20 s colonic distension stimulus. Average response adaptation in all LF cells was 1 ± 0.1 Hz, significantly (p > 0.001) less than that seen in the HF population. However, percentage adaptation in LF cells [62 ± 4% (terminal firing rate/peak firing rate)] is similar to that seen in HF cells, which likely reflects the difference in firing rate and dynamic range. The adaptation differences between LF and HF cells are difficult to interpret without knowing the pattern of connections to second-order neurons and contribution(s) of each class of colon afferents to the overall perception of visceral pain.
An examination of the instantaneous action potential firing rate elicited by maximal (40 cmH2O) colon distension revealed two statistically distinct populations of colon afferents. Each population is a Gaussian distribution with a shared boundary at ∼10 Hz (Fig. 3). In subsequent analyses, the action potential response elicited by maximal (40 cmH2O) colon distension was used to classify cells as HF or LF colon afferents. Most colon afferents (89 of 127) were characterized as LF cells; 30% of colon afferents were characterized as HF cells. Interestingly, LF cells appeared to have elevated distension response thresholds to action potential firing (Fig. 2, compare A, B). In fact, most LF cells did not respond to the mildest distension used in this study (10 cmH2O), whereas this stimulus was sufficient to elicit action potential firing in nearly all HF cells. Most (36 of 38) HF cells had measured thresholds of 10 cmH2O distension, whereas most (70 of 90) LF cells had measured thresholds of 20, 30, 40, or 50 cmH2O distension.
To examine absolute distension thresholds in these two populations of neurons, we conducted regression analysis for each colon afferent using the linear region of the mean firing response to the distension series (Sengupta and Gebhart, 1994a). The calculated absolute distension response thresholds for all cells examined are shown in Figure 4A; measured minimal distensions (10, 20, 30 or 40 cmH2O) required to elicit unit response are also given (Fig. 4A, inset). Analysis of absolute threshold revealed that colon afferents that coded maximal colon distension with firing frequencies >10 Hz (HF cells) had mean response thresholds of 2.6 ± 0.4 cmH2O, whereas those cells with maximal firing frequencies <10 Hz (LF) had mean thresholds of 17.5 ± 1.1 cmH2O. Both measured (Fig. 4A, inset) and absolute (Fig. 4A) thresholds differ significantly between HF and LF colon afferents (p < 0.001). These data suggest that HF and LF cells may be further characterized as low (HF) and high (LF) threshold cells.
This difference in action potential firing pattern is not simply attributable to a difference in threshold between LF and HF afferents. Maximal instantaneous firing rates elicited by each distension pressure were determined to define the stimulus response function for LF and HF cells. These data are shown in Figure 4B. Firing frequencies recorded from HF and LF afferents were significantly different at each distension pressure (p < 0.001) (Fig. 4B). Interestingly, colon distensions to much higher pressures (>50 cmH2O) did not elicit action potential firing in LF afferents comparable with firing rates recorded in HF afferents (data not shown), suggesting that LF cells are functionally distinct from HF cells. We propose that distension-sensitive colon afferents are functionally divided into two classes: HF cells, characterized by high firing rates, low response thresholds, and brief action potential durations; and LF cells, characterized by low firing rates, high response thresholds, and longer action potential durations.
Physiological characteristics of TRPV1-positive cells
A total of 52 colon afferents characterized by distension response was subsequently Neurobiotin-filled and recovered for immunohistochemical analysis. Cells were examined for expression of the capsaicin receptor, TRPV1, a nonselective ion channel shown previously to define a subset of nociceptors and implicated in heat and inflammatory pain (Caterina et al., 1997, 2000). Expression of GFRα3, the specific receptor for artemin, a member of the GDNF (glial cell line-derived neurotrophic factor) family of growth factors (Baloh et al., 1998), was also examined in colon afferents. We previously showed that artemin potently sensitizes TRPV1 channels in vitro and in vivo (Malin et al., 2006). Thirty-two cells were TRPV1-positive, and 20 were TRPV1-negative (Fig. 2, Table 1). GFRα3 was not observed in any TRPV1-negative neuron; however, 50% of the TRPV1-positive afferents also expressed GFRα3.
Most (86%) LF afferents expressed TRPV1 immunoreactivity, but few (13%) HF afferents were TRPV1 positive; these differences were highly significant (p < 0.001). Consistent with this correlation between LF/HF cell type and TRPV1 expression, most (29 of 32) TRPV1-positive afferents had higher distension thresholds and responded to 20, 30, 40, or 50 cmH2O distension, whereas most (17 of 20) TRPV1-negative afferents responded to the lowest distension pressures (5 or 10 cmH2O), as well as to more noxious stretch stimuli.
Both absolute (Fig. 5A) and measured (Fig. 5A, inset) distension response thresholds were significantly (p < 0.001) higher in TRPV1-expressing afferents (TRPV1-positive, 12.5 ± 1.4 cmH2O; TRPV1-negative, 3.6 ± 0.8 cmH2O). In addition, TRPV1-expressing colon afferents respond to colon distension with significantly (p < 0.001) lower rates of action potential firing than do TRPV1-negative afferents at all pressures examined (at 40 cmH2O: TRPV1-positive, 7.3 ± 1.3 Hz; TRPV1-negative, 21.7 ± 2.1 Hz) (Fig. 5B). Seventy-five colon afferents were physiologically characterized but not immunohistochemically identified (Table 1). Sixty-five percent would be categorized as LF afferents based on the action potential firing response to maximal colon distension (Fig. 5B). Interestingly, this is the same percentage of colon afferents that have been demonstrated to express TRPV1 in immunohistochemical studies (Christianson et al., 2006b). Together, these data suggest that TRPV1 expression occurs primarily in LF afferents.
Sensitization of colon afferents by protons, capsaicin, and mustard oil
In experiments in isolated sensory neurons, we and others have reported that direct application of capsaicin or mustard oil induces action potential firing and/or calcium influx in subsets of sensory neurons (Jordt et al., 2004; Elitt et al., 2006; Akopian et al., 2007). The action(s) of capsaicin and mustard oil are highly correlated to expression of TRPV1 and TRPA1 receptors, respectively. To examine the response properties of intact colon afferents to these compounds, the lumen of the colon was infused with capsaicin and mustard oil at concentrations that, in isolated neurons, have previously been demonstrated to produce receptor-specific responses (Caterina et al., 2000; Jordt et al., 2004; Malin et al., 2006; Akopian et al., 2007). In addition, we tested responses to low pH, pH 4, because protons are known to activate sensory neurons via acid-sensitive channels, such as TRPV1, and members of the acid-sensitive ion channel (ASIC) family. Functional expression of both TRPV1 and ASIC family members has been demonstrated in colon afferents (Jones et al., 2005; Page et al., 2005; Christianson et al., 2006a,b; Hughes et al., 2007; Sugiura et al., 2007).
In most cases, an individual cell was tested with more than one stimulus (Table 1). Agonists were given for 5 min, during which time firing activity was recorded to identify direct agonist responses. Then a colon distension series (10, 20, 30, and 40 cmH2O) was applied to evaluate sensitization of distension responses by each agonist. Drugs were washed out with ACSF or vehicle, as appropriate. Only when responses returned to baseline pretreatment levels were additional agonists applied. In addition, stimulus order was varied between cells, and agonist responses did not correlate with the order of drug delivery (see Materials and Methods). Representative data are shown in Figure 6. The colon afferent in the top panel showed sensitization of responses to colon distension after capsaicin, mustard oil, and acid, with complete washout in between application of each drug. (Note that the direct action potential firing responses to each agonist are not shown here.) Importantly, 5% DMSO, the vehicle for both capsaicin and mustard oil in these experiments, had little effect on response to colon distension. The cell in the bottom panel also shows little effect of DMSO, but pronounced sensitization of distension responses in capsaicin and low pH. Interestingly, this cell was not sensitized by mustard oil, suggesting that the overlap between TRPV1 and TRPA1 function, although significant, is not complete in colon afferents.
Effect of sensitizing agents on colorectal compliance
Biomechanical properties of the colon might be altered during repeated distension and in response to drug application; the following experiment was performed to address this potential confound. Pressure–volume curves were obtained from four colons tested with repeated distension series in the presence and washout of each agonist. Each colon was tested four times during each agonist and washout to obtain average pressure and volume data. Each colon was tested with six solutions. The sequence of agonist application was identical with that used during a typical electrophysiological experiment. Volume needed to maintain constant colon pressure could be resolved to 0.001 ml; data are shown in Table 2. The volume required to maintain the each pressure was not affected by the presence of any of the agonists or vehicles used in this study. These results indicate that changes in biomechanical properties of the colon were not responsible for the observed changes in neuronal response properties reported in this study.
Actions of capsaicin, mustard oil, and low pH are restricted to TRPV1-expressing neurons
Colon afferents were classified as responders for a given agonist if, after agonist application, cells fired action potentials or showed an increase (more than twofold of that seen before drug application) in ongoing activity (Table 1). Neurons were classified as “sensitized” if no direct firing response to drug application was seen, but if cells did show enhanced responses to subsequent colon distension (Table 1). Our analysis shown in Figure 5B demonstrated that TRPV1 is expressed by the majority of LF afferents. Previous studies have suggested that TRPA1 is expressed in a subset of TRPV1-expressing sensory neurons (Bandell et al., 2004; Jordt et al., 2004). Based on these observations, we expected capsaicin and mustard oil responses primarily in LF colon afferents. However, although more LF than HF cells responded to capsaicin and acid, these differences were not significant, probably because of the small number of cells tested. However, mustard oil sensitized LF significantly more often than HF neurons.
All of the pharmacologically tested neurons were also examined for TRPV1 expression. All cells “responding” and/or “sensitized” by mustard oil, capsaicin, or acid expressed TRPV1. Capsaicin, mustard oil, and acid application to the colon each induced significant direct responses in the majority of TRPV1-expressing afferents (in Hz: CAP, 5.0 ± 2; MO, 5.5 ± 2; acid, 3.3 ± 2) (Table 1). Only one cell classified as a “responder” was spontaneously active before drug application (2.2 Hz). Direct responses to TRPV1 and TRPA1 agonists were not seen in TRPV1-negative afferents. The majority of these affected cells were LF afferents. Interestingly, the few LF afferents that were not modulated by capsaicin, mustard oil, and low pH did not express TRPV1. TRP agonist modulation was seen in a few HF afferents. Three of nine HF afferents exposed to capsaicin exhibited both de novo action potential firing and increased responses to colon distension. Two of these cells were also activated and sensitized by mustard oil and one was activated and sensitized by low pH. All three of these cells expressed TRPV1.
Absolute colon distension thresholds calculated before and after agonist application for all cells are given in Figure 7, left. Capsaicin, mustard oil, and low pH reduced absolute colon distension thresholds in nearly all TRPV1-expressing afferents. Importantly, DMSO, the vehicle for capsaicin and mustard oil delivery, did not alter absolute colon distention thresholds (Fig. 7, bottom left). TRPV1-expressing afferents showed significant sensitization to colon distention after capsaicin application. This was evident at all distension pressures (Fig. 7, right). These data suggest that the effects of capsaicin recorded in this study are direct effects on sensory afferents and do not arise from TRPV1 expression in enteric neurons or non-neuronal structures of the colon. Mustard oil also sensitized responses to colon distension in TRPV1-expressing afferents at higher distension pressures (>10 cmH2O) (Fig. 7). As with capsaicin, mustard oil effects were also restricted to TRPV1-expressing colon afferents. This result suggests that TRPA1 is expressed in most TRPV1-expressing cells. Low pH effects were less dramatic that those seen after capsaicin or mustard oil application (Fig. 6, compare capsaicin and acid responses), but sensitization was significant (p < 0.001) at higher (>20 cmH2O) distension pressures (Fig. 7). Sensitizing effects of low pH were also restricted to TRPV1-expressing neurons. This result suggests that TRPV1-expressing neurons are the primary acid sensors for the colon.
Discussion
The primary goal of these studies was to characterize colon afferents using an approach that combined functional electrophysiology, immunohistochemistry, and pharmacology. We developed a novel preparation to record intracellularly from intact colon afferents, similar to that used by Bielefeldt et al. (2006) to characterize gastric afferents. In this study, we identified HF and LF colon afferents. HF afferents fired at high frequencies in response to colon distension and had low distension thresholds and significantly briefer action potential durations than LF cells. However, HF afferents were less numerous than HF cells. Interestingly, the HF and LF populations of colon afferents described here had many of the same properties described previously for low-threshold and high-threshold afferents, respectively. HF afferents in the present study are similar to low-threshold fibers in previous studies; LF afferents had high thresholds and low firing frequencies similar to previously described high-threshold afferents (Sengupta and Gebhart, 1994a; Gebhart, 1999; Rong et al., 2002; Daly et al., 2007).
We did note several differences between these results and previous studies of visceral afferents. First, despite recording from 127 colon afferents, we did not observe fibers conducting in the myelinated range (>1.0 m/s). This is contrast to previous reports in rats, in which ∼10% of colon afferents are Aδ fibers (Sengupta and Gebhart, 1994a). The differences between these results and the present study could be attributable to species differences. Second, in this study, we observed a relatively low number of spontaneously active colon afferents, whereas previous studies have reported significant numbers of fibers with ongoing activity. The reason(s) underlying these differences are not clear but may be attributable to species differences between rats and mice, different models of colon stimulation, and different electrophysiological preparations. Some previous studies have used balloon distension; the constant presence of the balloon in the colon may have elicited baseline afferent activation (Sengupta and Gebhart, 1994a). However, spontaneous activity was also common in a study of mouse jejunal afferents in which distension was produced by injection of fluid, as in this study. However, the recordings were made from teased fibers (compared with intact neurons in our study) and this may have contributed to the greater appearance of spontaneous activity (Rong et al., 2004). Lynn and Blackshaw (1999) also reported a greater number of spontaneously active fibers using a teased fiber preparation. However, in addition to the different recording site (axon vs soma), in the previous study the colon was opened along the longitudinal axis to allow stimulation of the mucosal surface. It is possible that this procedure sensitized or damaged afferent endings, producing the observed ongoing activity. The prevalence of spontaneous activity in vivo is of significant interest because ongoing activity has been proposed to underlie efferent effects of sensory fibers on the target tissue (Holzer, 2006).
Previous studies have reported that 20–25% of distension-responsive colon afferents have high response thresholds to colon distension (Sengupta and Gebhart, 1994a,b). In contrast, we report ∼65% of distension-responsive afferents are high-threshold low-firing rate fibers (LF). There are several possible reasons for this difference. First, the previous studies were performed in vivo and recordings were obtained from teased pelvic nerve fibers. In this study, recordings were obtained in an ex vivo preparation using intracellular electrodes to record from intact sensory neurons. Cutting and teasing the pelvic nerve may have altered the response properties of those afferents, compared with intact afferents. Second, the previous studies were performed in rats, whereas those reported here were performed in mice. Recently, we reported that the distribution and neurochemical phenotype of colon afferents in mice and rats is significantly different (Christianson et al., 2007), suggesting that there may be functional differences in colon afferents between these species as well. Finally, in our preparation, the mechanics of colon distension may be significantly altered from those reported in in vivo experiments. In the ex vivo preparation described here, it is likely that the colon responds to the same distension pressure with greater expansion and stretch than seen in situ, because the supporting pelvis and abdominal structures have been dissected away. This supranormal stretch may excite neurons normally silent, but recruited in injury or disease states. In support of this possibility, we found that LF afferents are activated and sensitized by TRP channel activation, suggesting that these cells are likely to be sensitized by inflammatory mediators, growth factors, and nucleotides (Malin et al., 2006, 2008; Dai et al., 2007; Andersson et al., 2008; Cruz-Orengo et al., 2008). This property suggests that some LF afferents in our study may be converted from silent afferents in vivo. Alternatively, in previous studies from other laboratories, the high proportion of HF fibers could be attributable, at least in part, to conversion of LF into HF fibers via sensitization by various stimuli. Thus, there may be continuum in response properties of LF/TRPV1-positive neurons; many may be silent until converted by noxious stimuli, at which point they assume the properties of LF or HF afferents depending on the intensity of noxious stimuli and/or environment.
Our initial emphasis on TRPV1 was based on a number of studies demonstrating that the majority of colon afferents are TRPV1-positive (Robinson et al., 2004; Brierley et al., 2005; Christianson et al., 2006a,b). In addition, previous studies of bladder and colon afferents suggested a functional role for TRPV1 in visceral mechanosensation. Altered micturation was reported in TRPV1−/− mice; however, because the TRPV1 channel is also expressed in the bladder urothelium, the role of TRPV1 in the primary afferent is unclear (Birder et al., 2002; Birder, 2006). Interestingly, both TRPV1−/− and ASIC3−/− mice have decreased visceromotor responses to colorectal distension (Jones et al., 2005). This result suggests that TRPV1 plays an important functional role in the response to colon distension, but the afferent populations affected (high or low threshold) by the removal of TRPV1 is unknown. Recent studies have revealed a requirement of TRPV1 expression for development of colon hypersensitivity in mice treated with intracolonic zymosan (Jones et al., 2007). Interestingly, zymosan treatment does not produce overt inflammation, but did cause long-lasting behavioral hypersensitivity. This suggests that, at least for visceral tissues, TRPV1 is necessary for hyperalgesia produced by both inflammatory and noninflammatory insults. Thus, TRPV1-expressing afferents may play a general role in plastic processes associated with persistent visceral pain states.
Although TRPV1 is expressed in hypersensitive afferents after inflammation, it is important to note that these afferents also express other channels and receptors shown previously to contribute to hypersensitivity. In mice, most TRPV1-positive afferents also express TRPA1, and like TRPV1, ablation of TRPA1 reduces development of inflammatory hyperalgesia (Bautista et al., 2006; Kwan et al., 2006). In addition, 80% of TRPV1-positive fibers express receptors for NGF (trkA) (Orozco et al., 2001) and ∼90% express the specific receptor for artemin (GFRα3) (Orozco et al., 2001; Malin et al., 2006). Expression of these growth factors has been shown to increase in inflamed tissues (Weskamp and Otten, 1987; Donnerer et al., 1992; Woolf et al., 1994; Malin et al., 2006) and injection of these growth factors alone produces significant prolonged hypersensitivity (Lewin et al., 1994; Dyck et al., 1997; Malin et al., 2006). Therefore, the apparent requirement for TRPV1 in inflammatory pain may be related to the function of the neurons that express this channel, as well as to the context of TRPV1 expression with other receptors and channels in the plasma membrane.
Footnotes
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This work was supported by National Institutes of Health Grants NS31826 and NS050758 (B.M.D.) and DK080182 (J.A.C.). We thank Chris Sullivan for outstanding technical support and Dr. H. R. Koerber for invaluable consultations regarding the development of the ex vivo preparation.
- Correspondence should be addressed to Brian M. Davis, Department of Medicine, Division of Gastroenterology, Hepatology, and Nutrition, E1440 Basic Medical Science Tower, University of Pittsburgh School of Medicine, 200 Lothrop Street, Pittsburgh, PA 15261.
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