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.

1. This study was performed to elucidate the actions of 5_hydroxytryptamine (5_HT) on mesenteric afferent discharge and to determine the receptor-mechanisms responsible for these effects. The activity of mesenteric afferents innervating the mid-jejunum of urethaneanaesthetized rats was recorded with extracellular microelectrodes. The discharge of single nerves within the whole nerve recording was monitored using waveform discriminator software. 2. The intravenous injection of 5_HT produced a complex pattern of afferent activation with two distinct components which could be distinguished both in terms of the response characteristics and the receptors involved. Initially, in 64% of nerve bundles, there was a brief (2·0 ± 0·1 s) but intense activation of afferent discharge with peak afferent firing increasing with incremental doses of 5_HT. The discharge frequency in seventeen single units from these bundles during the initial response to 10 ìg 5_HT was 13·0 ± 1·8 impulses s¢ from a baseline discharge of 1·0 ± 0·1 impulses s¢. 3. This initial response was mimicked by the 5_HT× receptor agonist, 2-methyl-5_HT, whereas 5_methoxytryptamine (5_MEOT, 10-100 ìg) had no comparable effect. Similarly, the initial 5_HT response was completely abolished by the 5_HT× receptor antagonist, granisetron (0·5 mg kg¢). 4. 5_HT also evoked, in approximately 35% of nerve bundles, a delayed response that single unit analysis showed to be mediated by an entirely different population of afferents from those activated during the initial response. This secondary response to 5_HT was characterized by a more prolonged (> 30 s) but less intense period of afferent activity which was coincident with an increase in intrajejunal pressure, and was mimicked by 5_MEOT (10-100 ìg). 5. The secondary response to 5_HT and the response to 5_MEOT were significantly attenuated by the 5_HT2A receptor antagonist, ketanserin (0·5 mg kg¢), which had no effect on the initial response. 6. The initial response to 5_HT was unaffected by the L-type calcium channel inhibitor nifedipine (1 mg kg¢) or the N-type calcium channel inhibitor ù_conotoxin GVIA (25 ìg kg¢). However, the secondary response to 5_HT was significantly reduced after treatment with nifedipine. 7. These results demonstrate that 5_HT activates different populations of afferent fibres innervating the rat jejunum. One population of afferents is activated directly via stimulation of 5_HT× receptors, while another population responds to 5_HT with a time course consistent with secondary activation of mechanosensitive afferents following 5_HT2A-mediated contractile activity.

Myenteric afterhyperpolarizing (AH) neurons are primary afferent neurons within the gastrointestinal tract. Stimulation of the intestinal mucosa evokes action potentials (AP) that are followed by a slow afterhyperpolarization (AHP(slow)) in the soma. The role of intracellular Ca(2+) ([Ca(2+)](i)) and ryanodine-sensitive Ca(2+) stores in modulating the electrical activity of myenteric AH neurons was investigated by recording membrane potential and bis-fura-2 fluorescence from 34 AH neurons. Mean resting [Ca(2+)](i) was approximately 200 nM. Depolarizing current pulses that elicited APs evoked AHP(slow) and an increase in [Ca(2+)](i), with similar time courses. The amplitudes and durations of AHP(slow) and the Ca(2+) transient were proportional to the number of evoked APs, with each AP increasing [Ca(2+)](i) by approximately 50 nM. Ryanodine (10 microM) significantly reduced both the amplitude and duration (by 60%) of the evoked Ca(2+) transient and AHP(slow) over the range of APs tested (1-15). Calcium-induced calcium release (CICR) was graded and proportional to the number of APs, with each AP triggering a rise in [Ca(2+)](i) of approximately 30 nM Ca(2+) via CICR. This indicates that CICR amplifies Ca(2+) influx. Similar changes in [Ca(2+)](i) and AHP(slow) were evoked by two APs in control and six APs in ryanodine. Thus, the magnitude of the change in bulk [Ca(2+)](i) and not the source of the Ca(2+) is the determinant of the magnitude of AHP(slow). Furthermore, lowering of free [Ca(2+)](i), either by reducing extracellular Ca(2+) or injecting high concentrations of Ca(2+) buffer, induced depolarization, increased excitability, and abolition of AHP(slow). In addition, activation of synaptic input to AH neurons elicited a slow excitatory postsynaptic potential (sEPSP) that was completely blocked in ryanodine. These results demonstrate the importance of [Ca(2+)](i) and CICR in sensory processing in AH neurons. Activity-dependent CICR may be a mechanism to grade the output of AH neurons according to the intensity of sensory input.

5_Hydroxytryptamine (5_HT) has widespread actions within the gastrointestinal tract with effects on both neural and non-neural target tissue. We have recently demonstrated that 5_HT stimulates mesenteric afferent nerve bundles supplying the rat jejunum via two different 5_HT receptor subtypes which appear to be located on two distinct populations of neurones innervating the intestine (Hillsley, Kirkup & Grundy, 1998). The most dramatic action of 5_HT is due to the stimulation of 5_HT× receptors since the 5_HT response can be mimicked by the 5_HT× receptor agonist 2_methyl-5_HT and abolished by the 5_HT× receptor antagonist granisetron. This is likely to be a direct action on the afferent nerve terminal since the response to 5_HT persisted after treatment with L_ and N-type calcium channel blockers (Hillsley et al. 1998) However, in approximately one-third of mesenteric bundles, this initial response was followed by a secondary response to 5_HT that was mediated by 5_HT2A receptors and attenuated by nifedipine, which also blunted the mechanical response to 5_HT (Hillsley et al. 1998 1. This study was performed to elucidate the type of afferents that mediate the multiple actions of 5_hydroxytryptamine (5_HT) on mesenteric nerve discharge. Electrophysiological recordings were made from mesenteric afferents innervating the mid-jejunum of the urethane-anaesthetized rat. The discharge of single nerves within the whole nerve recording was monitored using waveform discrimination software. 2. Afferents responded to 5_HT in one of two ways: a short latency, transient excitation mediated by 5_HT× receptors, or a delayed onset, more prolonged effect that was 5_HT2A receptor mediated. Afferents showing the 5_HT×-mediated response did not respond to luminal distension but were sensitive to intraluminal hydrochloric acid (150 mÒ) in twentyeight of twenty-nine experiments. In eight experiments, the 5_HT×-mediated response was reversibly abolished by a 2 min exposure to intraluminal application of local anaesthetic (2 % Xylocaine). 3. Mechanosensitive afferents which responded to distension (< 10 cmHµO) did not show a 5_HT×-mediated response (P = 0·92, n = 14), and maintained this mechanosensitivity after luminal anaesthesia. Mechanosensitive afferents did show a secondary response to 5_HT that was significantly attenuated by atropine (100-200 ìg kg¢), whereas hexamethonium (8 mg kg¢) had no effect. 4. In animals whose vagal afferent contribution to their mesenteric nerves had been eliminated by chronic truncal vagotomy, the 5_HT×-mediated response was absent in thirty-six of thirty-six nerve bundles. In contrast, mechanosensitivity to distension and the secondary response to 5_HT could still be evoked. 5. These results suggest that 5_HT stimulates mesenteric afferents by a direct action on 5_HT×receptors that are present on vagal mucosal afferent terminals. The mucosal afferent response to luminal acid, however, was unaffected by treatment with granisetron (0·5 mg kg¢) indicating that endogenous 5_HT from enterochromaffin cells...

Tetrodotoxin-resistant (TTX-R) sodium currents have been proposed to underlie sensory neuronal hyperexcitability in acute inflammatory models, but their role in chronic models is unknown. Since no pharmacological tools to separate TTX-R currents are available, this study employs Na v 1.8 and Na v 1.9 null mice to evaluate these currents roles in a chronic hyperexcitability model after the resolution of an inflammatory insult. Transient jejunitis was induced by infection with Nippostrongylus brasiliensis (Nb) in Na v 1.9 and Na v 1.8 null, wild-type and naïve mice. Retrogradely labelled dorsal root ganglia (DRG) neurons were harvested on day 20-24 post-infection for patch clamp recording. Rheobase and action potential (AP) parameters were recorded as measures of excitability, and Na v 1.9 and Na v 1.8 currents were recorded. DRG neuronal excitability was significantly increased in post-infected mice compared to sham animals, despite the absence of ongoing inflammation (sham = 1.9 ± 0.3, infected = 3.6 ± 0.7 APs at 2× rheobase, P = 0.02). Hyperexcitability was associated with a significantly increased amplitude of TTX-R currents. Hyperexcitability was maintained in Na v 1.9 -/-mice, but hyperexcitability was absent and APs were blunted in Na v 1.8 -/-mice. This study identifies a critical role for Na v 1.8 in chronic post-infectious visceral hyperexcitability, with no contribution from Na v 1.9. Nb infection-induced hyperexcitability is not observed in Na v 1.8 -/-mice, but is still present in Na v 1.9 -/-mice. It is not clear whether hyperexcitability is due to a change in the function of Na v 1.8 channels or a change in the number of Na v 1.8 channels.