Excitation of rat colonic afferent fibres by 5-HT(3) receptors

Abstract

The gastrointestinal tract contains most of the body's 5-hydroxytryptamine (5-HT) and releases large amounts after meals or exposure to toxins. Increased 5-HT release occurs in patients with irritable bowel syndrome (IBS) and their peak plasma 5-HT levels correlate with pain episodes. 5-HT(3) receptor antagonists reduce symptoms of IBS clinically, but their site of action is unclear and the potential for other therapeutic targets is unexplored. Here we investigated effects of 5-HT on sensory afferents from the colon and the expression of 5-HT(3) receptors on their cell bodies in the dorsal root ganglia (DRG). Distal colon, inferior mesenteric ganglion and the lumbar splanchnic nerve bundle (LSN) were placed in a specialized organ bath. Eighty-six single fibres were recorded from the LSN. Three classes of primary afferents were found: 70 high-threshold serosal afferents, four low-threshold muscular afferents and 12 mucosal afferents. Afferent cell bodies were retrogradely labelled from the distal colon to the lumbar DRG, where they were processed for 5-HT(3) receptor-like immunoreactivity. Fifty-six percent of colonic afferents responded to 5-HT (between 10(-6) and 10(-3) M) and 30 % responded to the selective 5-HT(3) agonist, 2-methyl-5-HT (between 10(-6) and 10(-2) M). Responses to 2-methyl-5-HT were blocked by the 5-HT(3) receptor antagonist alosetron (2 x 10(-7) M), whereas responses to 5-HT were only partly inhibited. Twenty-six percent of L1 DRG cell bodies retrogradely labelled from the colon displayed 5-HT(3) receptor-like immunoreactivity. We conclude that colonic sensory neurones expressing 5-HT(3) receptors also functionally express the receptors at their peripheral endings. Our data reveal actions of 5-HT on colonic afferent endings via both 5-HT(3) and non-5-HT(3) receptors.

Figure 1. Three types of fibre were classified on the basis of their responses to mechanical stimuli

In A, B, and C upper traces show instantaneous frequency and lower traces show raw electrophysiological data. A, a serosal receptor is activated only by probing the tissue (horizontal bars). B, a muscular receptor is activated by probing and by a maintained circular stretch of 8 mm; the response adapts rapidly during the stimulus (horizontal bars). No increase above baseline activity occurred in response to mucosal stroking. C, a mucosal receptor responds to probing and to stroking of its receptive field with a von Frey hair giving a force of 0.1 mN. D, the majority of fibres encountered were serosal receptors (grey segment; numbers shown around pie chart), the remainder being split between muscular (open segment) and mucosal receptors (filled segment). E, the distribution of receptive fields was concentrated near the mesenteric border and on the mesentery itself. Circles, serosal afferents; squares, muscular afferents; triangles, mucosal afferents; filled symbols indicate responsive to 5-HT agonists, open symbols indicate unresponsive. LSN/IMN, lumbar splanchnic nerve/intermesenteric nerve; IMG, inferior mesenteric ganglion; LCN, lumbar colonic nerves.

Figure 2. Colonic primary afferents were activated by 5-HT

A, B and C show examples of afferent responses to 5-HT application to the receptive fields of serosal, muscular and mucosal afferents, respectively (thick horizontal bars). Upper panels show spike frequency histograms (5 s bin width) of single unit activity. Lower panels show raw electrophysiological data. In B, the unit of interest is the one with the larger spike amplitude. D, concentration-response function of 7 serosal afferents to 5-HT. Estimated EC₅₀ was 2.93 × 10⁻⁶m. E, the number of fibres responsive to 5-HT (≥100 μm, filled segment, numbers adjacent), vs. unresponsive (open segment).

Figure 3. Colonic primary afferents were activated by the selective 5-HT₃ receptor agonist 2-methyl-5-HT

Legend for A, B and C as for Fig. 2. Spikes are per 5 s bin. Fibres recorded in A, B and C are the same as those in Fig. 2A-C. The muscular afferent in B did not show a reproducible increase in discharge, so the discharge shown was not considered as a response. D, concentration-response function of 6 serosal afferents to 2-methyl-5-HT. Estimated EC₅₀ was 2.83 × 10⁻⁵m. E, the number of fibres responsive to 2-methyl 5-HT (1 mm, filled segment, numbers adjacent), vs. unresponsive (open segment).

Figure 4. Inhibition of responses to 5-HT receptor agonists by the selective 5-HT₃ receptor antagonist alosetron

A, response of a serosal receptor to 100 μm 5-HT (horizontal bar) alone (i) and in the presence of 200 nm alosetron (ii). B, response of a serosal receptor to 1 mm 2-methyl-5-HT alone (i) and in the presence of 200 nm alosetron (ii). Note that response to probing (arrows) is unaffected. Spikes are per 5 s bin. C shows group data on effects of alosetron on responses to 5-HT and 2-methyl-5-HT. Both effects were significant (P < 0.05, n = 5). Maximal responses to 5-HT and 2-methyl 5-HT were not significantly different, although a trend was evident (P = 0.057).

Figure 5. 5-HT₃ receptors are present on a proportion of dorsal root ganglion (DRG) neurones

A, distribution of retrogradely labelled cells according to spinal segment. Data are expressed as mean number of labelled cells per section at each level. B, low- and medium-power fluorescence micrographs of L1 DRG showing 5-HT₃ receptor immunohistochemistry in green (FITC) and retrograde label in blue (Fast Blue). Colocalization is indicated by the arrows in vertically paired images of the same section. C, pie chart showing percentages of retrogradely labelled cells immunoreactive for 5-HT₃ receptors (green) vs. those lacking 5-HT₃ receptor immunoreactivity (blue) in eight DRG from four animals. D, cell size distribution of 5-HT₃ receptor-positive cells and retrogradely labelled cells.