Jejunal afferent nerve sensitivity in wild-type and TRPV1 knockout mice

Abstract

The aim of this study was to investigate the contribution of the TRPV1 receptor to jejunal afferent sensitivity in the murine intestine. Multiunit activity was recorded in vitro from mesenteric afferents supplying segments of mouse jejunum taken from wild-type (WT) and TRPV1 knockout (TRPV1^−/−) animals. In WT preparations, ramp distension of the gut (up to 60 mmHg) produced biphasic changes in afferent activity so the pressure–response curve had an initial rapid increase in afferent discharge followed by a second phase of slower increase in activity. Afferent response to distension was significantly lower in TRPV1^−/− than in WT mice. Single-unit analysis revealed three functional types of afferent fibres: (1) low-threshold fibres (2) wide dynamic range fibres and (3) high-threshold fibres. There was a marked downward shift of the pressure–response curve for wide dynamic range fibres in the TRPV1^−/− mice as compared to the WT controls. The afferent response to intraluminal hydrochloric acid (20 mm) was also attenuated in the TRPV1^−/− mice. In contrast, the response to bath application of bradykinin (1 μm, 3 ml) was not significantly different between the two groups. The TRPV1 antagonist capsazepine (10 μm) significantly attenuated the nerve responses to distension, intraluminal acid and bradykinin, as well as the spontaneous discharge in WT mice. The WT jejunal afferents responded to capsaicin with rapid increases in afferent activity, whereas TRPV1^−/− afferents were not at all sensitive to capsaicin. Previous evidence indicates that TRPV1 is not mechanosensitive, so the results of the present study suggest that activation of TRPV1 may sensitize small intestinal afferent neurones.

An extensive network of extrinsic and intrinsic sensory neurones innervates the gastrointestinal (GI) tract (Furness et al. 1998; Grundy, 2002). Stimulation of the extrinsic afferent neurones by mechanical stretch or chemical irritation results in reflex changes in gut functions, and in some circumstances, conscious sensations such as fullness, discomfort and pain. The precise signal transduction mechanisms by which various physical and chemical stimuli excite the gut afferents remain unclear. However, the potential for research in this field has exploded in recent years because of rapid progress in identifying molecules with potential sensory functions. Several ion channels including the P2X ATP receptors, acid-sensing ion channels and TRPV1 receptors have been identified and their roles in peripheral sensory processes have been explored (for reviews see Caterina & Julius, 1999; Gunthorpe et al. 2002; North, 2002; Krishtal, 2003).

The transient receptor potential channel TRPV1 (formerly known as VR1) was first cloned in 1997 by Caterina et al. (Caterina et al. 1997). TRPV1 is activated by heat, protons and vanilloid ligands such as capsaicin or endovanillioids such as anandamide (Caterina et al. 1997; Di Marzo et al. 2002). In addition, evidence is accumulating that protein kinases, including PKA and PKC, may directly activate or potentiate TRPV1 receptor (Premkumar & Ahern, 2000; De Petrocellis et al. 2001; Crandall et al. 2002).

TRPV1 receptor is predominantly expressed in small-diameter primary afferent neurones involved in nociception. Its role in somatic pain has been studied extensively. TRPV1 antagonists have been found to have antihyperalgesic activity in animal models of chronic inflammatory and neuropathic pain (Garcia-Martinez et al. 2002; Pomonis et al. 2003; Walker et al. 2003). TRPV1 knockout mice exhibit deficits in thermal hyperalgesia that accompany tissue injury and inflammation (Caterina et al. 2000; Davis et al. 2000).

TRPV1 receptor has also been identified on both intrinsic and extrinsic neurones of the GI tract (Anavi-Goffer & Coutts, 2003; Ichikawa & Sugimoto, 2003; Ward et al. 2003). Both vagal and spinal afferents of the GI tract can be activated by capsaicin (Su et al. 1999; Blackshaw et al. 2000). Patients with irritable bowel syndrome often exhibit an abnormal intolerance to normal gut stimuli (Accarino et al. 1995; Camilleri et al. 2001). A possible link between TRPV1 receptor and gut hypersensitivity has been implicated by the demonstration that TRPV1 receptor might be upregulated in inflamed and hypersensitive bowel in humans (Yiangou et al. 2001; Chan et al. 2003). However, there has as yet been no direct evidence that TRPV1 receptor is involved in gut sensitivity in normal or pathological situations.

To address these issues, we investigated the possible differences in small intestinal afferent nerve sensitivity between wild-type (WT, TRPV1^+/+) and TRPV1 knockout mice (KO, TRPV1^−/−) using an in vitro jejunum preparation. We found that TRPV1 KO mice had an attenuated afferent sensitivity to jejunal distension and intraluminal acid, suggesting that TRPV1 receptor is involved in modulating the gut sensitivity.

Methods

Animals

TRPV1 WT and KO mice with a genetic background of C57/BL6 were generated in GlaxoSmithKline (Harlow, UK). Transmembrane domains 2–4 of the mouse VR1 gene (i.e. DNA encoding amino acids 460–555) were replaced by the neo gene (for detail of target strategies, see Davis et al. 2000). Mating pairs of TRPV^−/− and TRPV1^+/+ N1F1 littermates were obtained to generate separate colonies of TRPV1 WT and KO mice at the University of Sheffield according to the UK Animals Scientific Procedures Act (1986). In the course of the study, genotyping was performed on randomly selected animals (six WT and six KO mice) to confirm the absence of TRPV1 gene in the KO mice. Twenty-six WT and 25 KO mice of either sex (8–12 weeks old) were used for nerve recording experiments. There were no overt differences in feeding behaviour, litter size, growth rate and body weight (24.8 ± 0.6 and 24.5 ± 0.6 g, P > 0.05) between WT and KO groups.

Tissue preparation

Mice were killed with an overdose of pentobarbitone sodium (80 mg kg⁻¹) applied intraperitoneally. A mid-line laparotomy was performed and sections of jejunum were removed between 10 and 20 cm rostral to the ileocaecal junction. Each piece of jejunum was between 30 and 35 mm in length, and was dissected out in such a manner that a non-bifurcating mesenteric bundle emanated centrally from each jejunal section. The intestinal segments were immediately placed into oxygenated (95% O₂+ 5% CO₂) Krebs solution (composition, mm: NaCl 120; KCl 5.9; NaH₂PO₄ 1.2; MgSO₄ 1.2; NaHCO₃ 15.4; CaCl₂ 2.5; glucose 11.5) at room temperature. One of the jejunal segments complete with mesenteric bundle was placed into a purpose-built chamber (20 ml). Krebs solution was constantly perfused through the tissue chamber (34 °C) at 10 ml min⁻¹ via a Minipuls 3 perfusion pump (World Precision Instruments (WPI), USA). The jejunum was cannulated at each end, and intraluminal pressure was recorded via a pressure amplifier (NL 108, Digitimer, UK). Two Genie syringe pumps (WPI) were connected in parallel to the intraluminal inflow cannula via a T-piece connector, to allow intraluminal perfusion of Krebs or different test solutions through the lumen. The mesenteric bundle was pinned out onto the Sylgard base of the recording chamber and a mesenteric nerve was carefully dissected from the bundle.

Nerve recording

Initially, conventional extracellular recording techniques were used, with mesenteric nerve mounted on a pair of platinum electrodes in a separate paraffin-filled chamber, as has been previously reported using rat tissue (Maubach & Grundy, 1999). However, in our hands the viability of this preparation with mouse tissue was limited to 1–2 h maximum. Therefore we switched to a suction electrode technique previously described for recordings from pelvic afferents of the in vitro mouse urinary bladder (Rong et al. 2002). Suction recording extended preparation viability and allowed for single-unit discrimination because of an enhanced signal to noise ratio. Nerve activity was recorded with a Neurolog headstage (NL100, Digitimer), amplified (NL104) and filtered (NL 125, band pass 200–3000 Hz). Whole-nerve activity was displayed on an oscilloscope (Tektronix TDS 210). The nerve signal was acquired (20 kHz sampling rate) to a computer through a Micro 1401 interface and Spike2 software (Cambridge Electronic Design, UK). Intraluminal pressure was sampled at 100 Hz. Single-unit discrimination was performed using the spike-sorting function of the Spike2 software (version 5.03).

Experimental protocols

The preparation was allowed to stabilize for up to 60 min. Throughout the experiment, Krebs solution was constantly perfused intraluminally at 10 ml h⁻¹ against a distal pressure head of 2 cmH₂O. Unless the gut was to be distended, the outlet cannula was kept open to allow free drainage so that any contractions would dispel content rather than raise intraluminal pressure. The recording configuration was set up in this way so as to minimize afferent activation secondary to variation in intraluminal pressure. To distend the intestine, a three-way tap on the intraluminal outlet cannula was closed whilst Krebs solution perfusion continued. In this manner, the gut segment was distended to 60 mmHg in approximately 120 s before the three-way tap was opened to return intraluminal pressure to baseline. This was repeated at regular intervals of 15 min to test the reproducibility of the nerve response to ramp distension, and the response during different test conditions. To test the effect of intraluminal hydrochloric acid (HCl), the perfusate was switched from Krebs solution to various concentrations of HCl (1–50 mm) in Krebs solution for 2 min. A minimum interval of 20 min was left between each HCl test. All other drugs were added to the Krebs solution perfusing the tissue chamber at a rate of 10 ml min⁻¹. Capsaicin (1–10 μm, 3 ml) and bradykinin (1 μm, 3 ml) were applied to the bath over a 20 s period.

Drugs

Capsaicin and capsazepine (Tocris Cookson, Avonmouth, UK) were dissolved in dimethylsulphoxide (DMSO) as 10 mm stock solution and were diluted in Krebs solution to required concentrations before use. The maximum final bath concentration of DMSO was 0.1%. Vehicle controls were included in these experiments. Bradykinin (Sigma, Poole, UK) was dissolved in saline. All inorganic salts were purchased from BDH (Poole, UK).

Data analysis

The mean firing frequency (imp s⁻¹) was measured with a time constant of 30 s. The pressure–response curve for whole-nerve or single-unit activity was plotted using the X–Y plot function of the Spike2 program. To illustrate the time course and amplitude of the responses to HCl or bradykinin, rate histograms were plotted for 10 min after commencement of the treatment. Statistical analysis was performed using Graphpad Prism version 3.00 (Graphpad Software, San Diego, USA). Where appropriate, data are expressed as means ± s.e.m. and compared using paired or unpaired Student's t tests. Non-parametric data are compared using the Mann–Whitney U-test for two groups or the Kruskal–Wallis test for multiple groups. Curves are compared using an analysis of covariance (ANCOVA). A P value of less than 0.05 is considered as statistically significant.

Results

Spontaneous nerve activity

In the experimental conditions of the present study, the mesenteric afferents of the jejunum exhibited irregular spontaneous activity (Fig. 1). The mean level of spontaneous whole-nerve activity in WT (16.03 ± 2.87 imp s⁻¹, n = 19) appeared to be higher than in TRPV1^−/− mice (9.94 ± 1.84 imp s⁻¹, n = 18), although statistical significance was not reached (P = 0.087, Student's t test) because of large variations in spontaneous activity from one preparation to another and the relatively small sampling size. In all recordings, it was possible to distinguish the activity of several single units based on the spike waveforms (Fig. 1). A total of 139 and 147 single units were discriminated in the WT and the TRPV1 KO mice, respectively. The average spontaneous discharge rate of single units was significantly higher in WT (1.26 ± 0.06 imp s⁻¹) than in TRPV1 KO preparations (0.52 ± 0.04 imp s⁻¹, P < 0.01).

Figure 1. Spontaneous mesenteric nerve activity in the mouse in vitro jejunum preparation.

Top trace, typical whole-nerve signal with multiple action potentials of different size and shape. Single-unit activity can be discriminated based on the spike waveform as is illustrated in the bottom panels in which the action potentials of three units are superimposed in separate templates (Units 1–3). Middle traces (Units 1–3) are the waveform of each single unit.

Responses to ramp distension

Repeated ramp distensions at 15 min intervals were performed by infusion of saline into the jejunum to a peak pressure of 60 mmHg in order to activate a spectrum of mechanoreceptors. In WT preparations (n = 19), ramp distension produced a biphasic activation of afferent fibres, as represented in Fig. 2A. The first phase of the response was a rapid increase in afferent activity during the first 1 mmHg change in intraluminal pressure. Over this pressure range, the afferent activity increased by 17.1 ± 3.18 imp s⁻¹. The second phase of the response was a slower increase in afferent firing between 2 and 60 mmHg. It appears that the rate of increase in afferent activity escalated when the pressure reached 20 mmHg (Fig. 2B). Thus, the slope of the pressure–response curve was 0.69 ± 0.06 imp s⁻¹ mmHg⁻¹ between 2 and 20 mmHg and it increased to 0.92 ± 0.07 imp s⁻¹ mmHg⁻¹ between 20 and 60 mmHg.

Figure 2. Mesenteric afferent responses to jejunal distension in wild-type (WT) and TRPV1^−/− mice.

A, representative recording of distension-induced biphasic changes in afferent activity in a WT preparation. The bottom trace is an expanded view of the nerve activity around the time when distension started. Spikes of larger amplitudes are truncated in order to illustrate spikes of lower amplitudes, which tend to be low-threshold fibres firing vigorously at low distension pressure. B, pressure–response relationships of multiunit activity in 19 WT and 18 TRPV1^−/− preparations. The curve is biphasic and the second phase can be further divided into two components (between 2 and 20 mmHg and between 20 and 60 mmHg). There are significant differences in the pressure–response curves between the WT and the TRPV1^−/− groups (ANCOVA, see text for detail). C, relationships between perfusion volume and intraluminal pressure of the jejunal segments are not different between the WT and the TRPV1^−/− groups.

Ramp distensions in TRPV1^−/− mice (n = 18) evoked very similar nerve response profiles to those observed in WT mice. The mean response was a biphasic activation of afferent nerve firing (Fig. 2B). However, the rate of increase in afferent activity was lower than in WT mice. Over the first 1 mmHg change in intraluminal pressure, the afferent activity increased by 13.74 ± 3.30 imp s⁻¹ (P = 0.47 compared to WT, Student's t test). The slope of the pressure–response curve was 0.31 ± 0.01 imp s⁻¹ mmHg⁻¹ (P < 0.01 compared to the WT, ANCOVA) between 2 and 20 mmHg, and 0.60 ± 0.05 imp s⁻¹ mmHg⁻¹ (P < 0.01 compared to the WT, ANCOVA) between 20 and 60 mmHg.

To investigate the potential difference in the compliance of the gut wall between WT and TRPV1^−/− mice, we plotted the relationship between volume and pressure of the jejunal segments, i.e. the average perfusion volume required to reach different levels of intraluminal pressure in the two groups (Fig. 2C). The two curves completely overlap each other, indicating that there was no marked difference in the mechanical property of the gut wall between WT and TRPV^−/− mice.

The pressure–response profiles of single units could distinguish three functional types of afferent fibres. The first type had a low threshold and its activity reached a peak rapidly in the first few millimetres of mercury rise of pressure. There was no further increase in discharge in these afferents with further increases in pressure up to 60 mmHg (Fig. 3A). Indeed in many cases, the activity dropped. These are typical of low-threshold fibres (LT fibres). The second type also had a low threshold for activation but the discharge gradually increased further with the rise of pressure (Fig. 3B). They were classified as low-threshold wide dynamic range fibres (WDR fibres) and their pressure–response relationship mimics that of the whole-nerve activity. The third type had a threshold at >20 mmHg (Fig. 3C). They were classified as high-threshold fibres (HT fibres). The relative distribution of these functional classes of afferents in WT and TRPV1^−/− mice was not different (Table 1). The pressure–response relationships of LT and HT fibres were comparable between WT and TRPV1^−/− mice. However, there was a significant shift of the pressure–response curve for WDR fibres in TRPV1^−/− as compared to that in TRPV1^+/+ mice. Thus, for the first 1 mmHg rise in pressure, the average increase in discharge rate of WDR fibres was 0.35 ± 0.04 imp s⁻¹ in the TRPV1^−/− and 0.73 ± 0.08 imp s⁻¹ in the TRPV1^+/+ mice, respectively (P < 0.001, Student's t test). In the TRPV1^+/+ mice, the slope of the curve was 0.033 ± 0.001 imp s⁻¹ mmHg⁻¹ between 2 and 20 mmHg, and 0.062 ± 0.002 imp s⁻¹ mmHg⁻¹ between 20 and 60 mmHg. In the TRPV1^−/− mice, the slope was 0.010 ± 0.002 imp s⁻¹ mmHg⁻¹ between 2 and 20 mmHg (P < 0.001 compared to WT, ANCOVA) and 0.040 ± 0.001 imp s⁻¹ mmHg⁻¹ between 20 and 60 mmHg (P < 0.001 compared to WT, ANCOVA).

Figure 3. Single-unit responses to jejunal distensions in WT and the TRPV1^−/− preparations.

A–C, pressure–response curves of three different functional types of fibres: low-threshold fibres (LT fibres), wide dynamic range fibres (WDR fibres) and high-threshold fibres (HT fibres). D, pressure–response curves of all fibres. Note that the curves for WDR fibres are significantly different between WT and TRPV1^−/− mice, but the curves for LT and HT fibres are not (ANCOVA).

Table 1.

Number of different types of mesenteric afferent fibres in wild-type (WT) and TRPV1 knockout (KO) mice

	TRPV1 WT	TRPV1 KO
Low-threshold fibres	19 (13.7%)	22 (15%)
Wide dynamic range fibres	94 (67.6%)	85 (57.8%)
High-threshold fibres	26 (18.7%)	40 (27.2%)
Total	139 (100%)	149 (100%)

P = 0.1808, χ² test.

The effects of the TRPV1 antagonist, capsazepine (CPZ), on the afferent responses to distension was investigated in TRPV1^+/+ mice. CPZ (3–30 μm, n = 3) inhibited the afferent responses to distension in a concentration-dependent manner, as is represented in Fig. 4A. In most experiments, only 10 μm CPZ was used. Following bath application of CPZ (10 μm), spontaneous discharge was decreased from 14.50 ± 1.97 to 8.41 ± 1.16 imp s⁻¹ (n = 10, P < 0.05, paired t test) and there was a marked shift of the pressure–whole-nerve response curve during distension (Fig. 4B). In the presence of CPZ, the mean increase in afferent discharge for the first 1 mmHg rise in pressure was 5.94 ± 1.31 imp s⁻¹ (vs. 9.42 ± 2.43 imp s⁻¹ in control, P < 0.05, paired t test). The slope of the curve was 0.37 ± 0.03 (vs. 0.53 ± 0.04 imp s⁻¹ mmHg⁻¹ in control, P < 0.05, ANCOVA) between 2 and 20 mmHg, and 0.57 ± 0.05 (vs. 0.97 ± 0.14 imp s⁻¹ mmHg⁻¹ in control, P < 0.01, ANCOVA) between 20 and 60 mmHg. Recovery of the afferent responses to distensions was observed after washing out the antagonist.

Figure 4. The effects of capsazepine (CPZ) on distension-induced mesenteric afferent activity in TRPV1 WT mice.

A, an example of changes in afferent discharge rate in response to increases in intraluminal pressure in control and in the presence of different concentrations of CPZ. This was a continuous recording in a TRPV1 WT preparation, but the traces are truncated for clarity of illustration. B, the pressure–multiunit response relationships in control and in the presence of CPZ 10 μm. The two curves are significantly different (ANCOVA).

The effects of CPZ on single-unit activity were analysed in a total of 68 units. As is illustrated in Fig. 5, all three types of afferent fibres were significantly inhibited by CPZ.

Figure 5. The effects of CPZ on single-unit responses to jejunal distensions.

A–C, pressure–response relationships of LT fibres, WDR fibres and HT fibres in control and in the presence of CPZ 10 μm. D, plots of the pressure–response curves of all single units averaged. CPZ resulted in a significant shift of the pressure–response curves of all three fibre types (ANCOVA).

Response to intraluminal acid

The proton sensitivity of jejunal afferents was assessed by intraluminal infusion of HCl diluted in Krebs solution for 2 min. In pilot experiments carried out in seven WT and seven TRPV1 KO mice, neither 1 mm nor 5 mm HCl had any significant effects on jejunal-nerve activity. In response to a 10–50 mm HCl infusion, however, both WT and TRPV1 KO jejunal-nerve preparations displayed clear excitatory responses in a concentration-dependent manner.

The afferent sensitivity to intraluminal application of 20 mm HCl (pH 4.8, 167 μl min⁻¹, 2 min) in the WT and TRPV1 KO preparations was systematically investigated. The afferent response to the first acid challenge was usually small, but the response became greater and stable after between two and three challenges. A typical trace of the afferent nerve activity following intraluminal infusion of HCl in the TRPV1 WT preparation is shown in Fig. 6A. The nerve activity started to increase after a latency of 30–75 s, reached a peak of 19.97 ± 1.89 imp s⁻¹ (n = 15) above the control level approximately 100–150 s after commencement of acid infusion, and then slowly recovered to the baseline over 10–20 min. Analysis of single-unit activity indicates that individual units responded to acid differently in terms of the latency, duration and magnitude of the response (Fig. 6A). As is illustrated in Fig. 7A, HT fibres tend to reach peak activity faster than LT and WDR fibres, the median value of time to peak being 119, 150 (P < 0.05, Kruskal–Wallis test) and 159 s (P < 0.01) for HT, WDR and LT fibres, respectively. Furthermore, the activity in LT fibres following acid infusion was much less compared to that seen in HT or WDR fibres (Fig. 7B).

Figure 6. The responses of jejunal afferents to intraluminal infusion of HCl in the WT and the TRPV1^−/− mice.

A, representative recording of the multiunit activity following intraluminal infusion of HCl (20 mm), with the bottom trace showing changes of the activity in six single units. B, comparison of the multiunit rate histogram following intraluminal acid infusion in WT (n = 12) and TRPV1^−/− preparations (n = 8). *P < 0.05 and **P < 0.01, Student's t test. C, the rate histogram of whole nerves following acid infusion in control and in the presence of CPZ in WT mice. *P < 0.05 and **P < 0.01, paired t test.

Figure 7. The response of single units in mesenteric nerves to intraluminal acid in TRVP1 WT mice.

A, frequency histogram of time to peak (the latency from the start of acid infusion to single-unit peak activity) in different functional types of fibres. The Kruskal–Wallis test indicates that HT fibres reached peak activity significantly faster than WDR fibres and LT fibres. B, the average rate histogram of different types of afferent fibres following application. Note that the response in LT fibres is less than in WDR and HT fibres.

In TRPV1^−/− mice (n = 11), the pattern of the afferent response to intraluminal acid is similar to that seen in TRPV1^+/+ mice. However, the amplitude of the response was smaller than in WT mice (Fig. 6B). The afferent discharge rate reached a peak of 11.20 ± 1.80 imp s⁻¹ above the control level (vs. 19.97 ± 1.89 imp s⁻¹ in the WT, P < 0.01). The level of afferent activity in the slow recovery phase did not differ significantly from that in the TRPV1^+/+ mice.

The effects of CPZ on the jejunal afferent nerve sensitivity to intraluminal acid was investigated in WT preparations (n = 12). CPZ (10 μm) significantly inhibited the acid responses. Before CPZ, the peak increase in discharge was 25.47 ± 4.03 imp s⁻¹. In the presence of CPZ, the peak increase in afferent activity was 15.61 ± 2.53 imp s⁻¹, which was significantly lower than that of the control response (P < 0.01, paired t test). Furthermore, the level of afferent activity in the recovery phase was also lower in the presence of CPZ (Fig. 6C).

Response to bradykinin

The afferent sensitivity to bradykinin was assessed in 13 WT and 14 TRPV1^−/− afferent preparations. Bath application of 1 μm bradykinin (3 ml, superfused over a 20 s period) induced a rapid increase in afferent discharge after a latency of about 30 s in WT preparations, and the activity returned to the baseline after 10–15 min, as represented in Fig. 8A. All three functional types of fibres responded to bradykinin. There was no significant difference in the magnitude or time course of the response among LT, WDR and HT fibres (Fig. 8B). Bradykinin treatment failed to induce significant alteration in intraluminal pressure, which might be because the lumen was constantly perfused with free drainage so that any contractions would dispel content rather than raise pressure.

Figure 8. The responses of mesenteric afferent nerve to bradykinin in WT and TRPV1^−/− mice.

A, representative recording of the multiunit afferent activity in response to bath application of bradykinin (BK 1 μm, 3 ml). B, the average-rate histogram of different types of afferent fibres following application of BK. C, comparison of the averaged level of increases in multiunit activity following application of BK (1 μm, 3 ml) in WT (n = 13) and in TRPV1^−/− preparations (n = 14). No significant difference was found between the two groups (Student's t test). D, the effects of CPZ on the multiunit responses to BK in seven TRPV1 WT preparations. *P < 0.05 and **P < 0.01, paired t test.

A similar pattern of changes in afferent activity was observed following application of bradykinin in the TRPV1^−/− preparations. There was no significant difference in the magnitude of the afferent response to bradykinin between TRPV1^−/− and TRPV1^+/+ preparations (Fig. 8C). However, in WT preparations (n = 7), CPZ (10 μm) inhibited the afferent response to bradykinin (Fig. 8D). In the presence of CPZ, the peak increase in afferent activity was 7.89 ± 3.49 imp s⁻¹, which was less than the control value of 14.87 ± 4.26 imp s⁻¹ (P < 0.01, paired t test).

Response to capsaicin

Bath application of capsaicin produced concentration-related changes in afferent nerve activity in the TRPV1^+/+ preparations. Brief exposure to 1 μm (3 ml) capsaicin elicited a short period of intense discharge (Fig. 9A). Exposure to increasing doses of capsaicin (3 and 10 μm) resulted in a brief excitation followed by a period of afferent desensitization characterized by reduced spontaneous discharge and profoundly attenuated responses to distension, bradykinin (Fig. 9B) and intraluminal acid (data not shown). In contrast, in TRPV1^−/− preparations (n = 10), capsaicin (1–10 μm, 3 ml) failed to induce an excitation of afferent fibres or desensitization (Fig. 9C).

Figure 9. The responses of mesenteric afferent nerves to capsaicin in WT and TRPV1^−/− mice.

A, upon administration of 1 μm capsaicin in WT mice, there was an excitation on mesenteric afferents. B, following 10 μm capsaicin, there was a brief phase of intense discharge followed by a decrease in spontaneous nerve firing and reduced responsiveness to BK, intraluminal acid (not shown) and distension, which is indicative of afferent nerve desensitization. C, cumulative data showing changes in afferent activity following application of capsaicin in WT preparations. Note that TRPV1 KO preparations failed to respond to capsaicin.

Discussion

This study is the first report of recordings from murine jejunal afferent nerves. Due to the inherent difficulties involved in performing prolonged in vivo recording in the mouse, we developed an in vitro approach to investigate the possible role of TRPV1 in the sensitivity of jejunal afferents to mechanical and chemical stimulations by using TRPV1-deficient mice. In WT preparations, capsaicin induced typical excitatory responses on the afferents, which lasted briefly and were followed by afferent desensitization with higher concentration of capsaicin (10 μm). In contrast, in TRPV1 KO mice, capsaicin had neither excitatory nor desensitizing effects on jejunal nerves. This demonstrates that the excitatory effects of capsaicin were indeed mediated by TRPV1 receptor and that TRPV1^−/− mice do not express functional ‘capsaicin receptors’ on jejunal afferent endings. We demonstrate that the loss of capsaicin sensitivity is accompanied by an attenuated afferent sensitivity to mechanical distension of the gut and to intraluminal acid infusion in the TRPV1 KO mice, suggesting that TRPV1 receptor may play an important role in the control of gut sensitivity.

In this preparation, the response to distension was similar to that seen in the rat in vivo (Booth et al. 2001). The afferents showed spontaneous discharge and responded to jejunal distension in a biphasic manner. The first phase was a rapid increase in discharge during the initial rise in intraluminal pressure. This was followed by a continuing but slower increase in afferent activity as the intraluminal pressure rose. These data are best explained by the activation of different types of mechanosensitive afferents. It has been proposed that in the rat the first rapid phase of response is largely due to the activation of low-threshold vagal afferents, whereas both spinal and vagal afferents contribute to the second phase of response (Booth et al. 2001). Single-unit analysis revealed three functional populations of afferent fibres with distinctive pressure–response characteristics: (1) those with a low threshold for activation and which fire maximally at low distension pressure (i.e. LT fibres); (2) those with a low threshold, but encoding across a wide range of intraluminal pressure and which make up the largest population of jejunal afferents (i.e. WDR fibres) and (3) those that only fire at a pressure above normal physiological range (>20 mmHg, HT fibres). The combined activity in these three populations gave rise to the biphasic whole-nerve response. However, it is important to note that, as with conventional recording methods using wire electrodes, the current multiunit suction recording does not differentiate vagal, spinal or intestinofugal fibres of enteric sensory neurones. These different sets of fibres play very different roles in local/systematic control of gut motility, secretion and perception.

TRPV1 receptor modulates jejunal afferent sensitivity to distension

TRPV1 receptors are not generally considered to be directly mechanically gated (Gunthorpe et al. 2002). The role of TRPV1 in the mechanical sensitivity in somatic afferents has been investigated extensively. TRPV1-null mutant mice had normal basal mechanical sensitivity and normal mechanical hyperalgesic responses following tissue inflammation but showed deficient basal thermal sensitivity and thermal hyperalgesia (Caterina et al. 2000; Davis et al. 2000). In contrast, pharmacological evidence suggests that TRPV1 is involved in mechanical hyperalgesia following tissue injury or inflammation. For examples, TRPV1 receptor antagonist, CPZ, reverses mechanical hyperalgesia in models of inflammatory and neuropathic pain (Walker et al. 2003). Earlier studies demonstrated that activation of TRPV1 by capsaicin leads to mechanical as well as thermal hyperalgesia in rat, primate and human subjects (Simone et al. 1989; LaMotte et al. 1992; Torebjork et al. 1992).

Several lines of evidence suggest that TRPV1 receptor may modulate jejunal afferent sensitivity to distension. Firstly, the multiunit afferent responses to distension were smaller in TRPV1^−/− than in WT preparations. Secondly, in WT animals, the afferent response to distension was inhibited by CPZ. Thirdly, acute desensitization of the afferents with capsaicin resulted in a profound reduction in afferent sensitivity in WT but not in TRPV1^−/− mice.

Of the three functional types of afferents, only WDR fibres showed attenuated mechanosensitivity in the TRPV1^−/− mice. The responses of LT and HT fibres were not significantly different between WT and TRPV1^−/− mice. Previous studies in rat suggested that LT fibres are mainly vagal afferents (Booth et al. 2001). TRPV1 immunoreactivity is weaker in nodose compared to dorsal root ganglion (DRG) neurones and TRPV1 immunoreactivity could not be detected on the vagal afferent terminal structures in the intestine that are believed to be mechanosensitive (Ward et al. 2003; Patterson et al. 2003). Therefore, both functional and morphological data would imply that the TRPV1 receptor is less important in mechanosensory transduction in vagal afferents in the small intestine. Interestingly, in TRPV1^+/+ mice, the responses of LT and HT fibres were attenuated by CPZ. CPZ has only modest potency for TRPV1 and is somewhat non-specific, also affecting voltage-sensitive calcium channels and nicotinic cholinergic receptors (Docherty et al. 1997; Liu & Simon, 1997). Therefore differences in the effects of genetic KO of TRPV1 from the effects of CPZ application may in part be explained by the non-specific actions of CPZ. However, it is also possible that compensatory changes in parallel signalling pathways during the development of TRPV1-null mutant mice may result in restored mechanosensitivity in LT and HT fibres.

How is TRPV1 receptor involved in mechanosensory transduction in jejunal afferent terminals? Perhaps the simplest explanation would be that TRPV1 receptor is mechanosensitive. However, although some TRP-related channels such as NompC, Nanchun and TRPV4 can be mechanically activated, until now there has been no direct evidence to support TRPV1 receptor can be mechanically gated.

An alternative explanation is that mechanical stimulation would result in the release of endogenous TRPV1 agonist(s), which in turn stimulates afferent terminals that express TRPV1 receptor. There is still some debate as to the nature of the endogenous ligands for TRPV1 receptor. Several arachidonate derivatives, including 12-lipoxygenase products (Hwang et al. 2000) and the endocannabinoid, anandamide (Zygmunt et al. 1999; Smart et al. 2000; Roberts et al. 2003), have been proposed as ‘endovanilloids’. More recently, N-arachidonoyl-dopamine and N-oleoyl-dopamine have been identified as potent endovanilloids, being even more potent than capsaicin at inducing hyperalgesia in vivo (Huang et al. 2002; Chu et al. 2003; Toth et al. 2003). However, there is as yet a paucity of information regarding if and how these putative TRPV1 ligands are involved in mechanosensory transduction, which was not investigated in this study, but will be an interesting subject for future research.

Considering the evidence for TRPV1's involvement in hyperalgesia associated with tissue injury or inflammation (Yiangou et al. 2001; Ji et al. 2002; Walker et al. 2003), a third possibility arises because the GI tract is continually exposed to antigenic or pathogenic material, some of which may be toxic and potentially hazardous elements of microbial or viral origin. In the GI tract, TRPV1 may be tonically activated by ‘endovanilloids’ and protons, or through second messenger systems involving PKA or PKC (Premkumar & Ahern, 2000; De Petrocellis et al. 2001; Crandall et al. 2002), which can be stimulated by inflammatory mediators such as bradykinin released as a result of the innate defence mechanisms. Consequently, TRPV1 receptor might be indirectly involved in mechanosensory transduction in that sensory fibres expressing TRPV1 receptor in the GI tract are pre-tuned to a certain level of excitability (i.e. sensitized). It follows that the jejunal afferents of TRPV1-deficient mice would have a lower excitability and would respond to mechanical stretch (and other stimuli) at a more reduced level than those of WT mice.

TRPV1 receptor contributes to jejunal afferent sensitivity to acid

The response of GI afferents to intraluminal HCl has been reported previously (Blackshaw & Grundy, 1990; Richards et al. 1996). The acid response was comprised of an acute rise in discharge followed by a prolonged recovery phase. The variations in the responses of single units to intraluminal acid might be attributable to the difference in the location of afferent terminals because fibres terminating in different layers of the gut wall were supposedly exposed to variable levels of acidity due to the diffusion barrier. LT fibres of the upper gut are known to terminate as specialized intraganglionic laminar endings in the myenteric plexus (Zagorodnyuk & Brookes, 2000; Zagorodnyuk et al. 2001). Less clear is the location of the terminals of WDR and HT fibres, at least some of which may terminate in the mucosa and submucosal region.

The whole-nerve response to acid was significantly lower in magnitude in the TRPV1^−/− than in WT preparations. Moreover, in WT mice, the responses to acid were significantly inhibited by CPZ. Similarly, CPZ reportedly inhibited acid-induced activity in airway afferent fibres in guinea pigs (Kollarik & Undem, 2002) and acid-induced writhing response in mice (Ikeda et al. 2001). In urethane-anaesthetized rats, CPZ dose dependently inhibited the hyperaemic response to acid and capsaicin, but did not affect bradykinin-induced hyperaemia in the duodenum (Akiba et al. 1999). These findings, in concert with previous observations in TRPV1 KO mice that DRG neurones or nerve fibres of the skin showed a marked reduction in proton-evoked responses (Caterina et al. 2000), suggest that TRPV1 is involved in jejunal afferent sensitivity to acid.

Protons have two main effects on TRPV1 receptor. First, TRPV1 can be activated at room temperature when the extracellular pH drops below 6. Second, protons potentiate the response to capsaicin or heat, and do so over a concentration range (pH 6.8) that matches the extent of local acidosis associated with various forms of tissue injury (Caterina et al. 1997). Apart from these direct mechanisms, TRPV1 might also be indirectly involved in acid sensitivity by pre-tuning the excitability of afferent terminals. Apparently, however, TRPV1 receptor does not solely account for the acid sensitivity since acid still evoked significant, albeit smaller increases in afferent discharge in TRPV1^−/− preparations. Several other molecules can potentially be involved in acid sensitivity, including acid sensing ion channels (Krishtal, 2003) and the P2X₂ ATP receptor (King et al. 1996). The importance of these receptors in acid sensitivity in the gut awaits further investigation.

TRPV1 is not essential for the afferent sensitivity to bradykinin

The jejunal afferent response to bath application of bradykinin in TRPV1^−/− mice was not significantly different from that in WT mice. In contrast, CPZ significantly inhibited the bradykinin response in WT preparations. It has been shown in the rat that the response to bradykinin is largely mediated by the BK₂ receptors (Maubach & Grundy, 1999). There has also been evidence suggesting that TRPV1 may be activated in response to BK₂ activation, a process involving PKC. For example, in the rat, CPZ largely inhibited bradykinin-induced C-fibre discharge in the skin–nerve preparations as well as bradykinin-evoked currents in DRG neurones (Shin et al. 2002). CPZ also inhibited bradykinin-induced discharges in vagal airway afferent fibres in guinea pigs (Carr et al. 2003). Treatment of cultured DRG neurones with bradykinin augments heat-evoked currents (Sugiura et al. 2002). However, the findings in this study suggest that TRPV1 is not essential for the afferent response to bradykinin.

In conclusion, this study demonstrates that the TRPV1 receptor plays an important role in controlling the jejunal afferent sensitivity to distension and acid in the mouse small intestine. Previous evidence indicates that TRPV1 is not mechanosensitive, so the results of the present study suggest that activation of TRPV1 may sensitize jejunal afferent neurones.