In vitro recordings of afferent fibres with receptive fields in the serosa, muscle and mucosa of rat colon

Abstract

1. Colonic afferent fibres were recorded using a novel in vitro preparation. Fibres with endings in the colonic mucosa are described, along with those in muscle and serosa, and their responses to a range of mechanical and chemical luminal stimuli. 2. Mechanical stimuli were applied to the tissue, which included stretch, blunt probing of the mucosa and stroking of the mucosa with von Frey hairs (10-1000 mg). Chemical stimuli were applied into a ring that was placed over the mechanoreceptive field of the fibre; these were distilled water, 154 and 308 mM NaCl, 100 microM capsaicin, 50 mM HCl, and undiluted and 50% ferret bile. 3. Recordings were made from 52 fibres, 12 of which showed characteristics of having endings in the mucosa. Mucosal afferents were sensitive to a 10 mg von Frey hair and were generally chemosensitive to >= 1 chemical stimulus. 4. Ten fibres showed characteristics of having receptive fields in the muscular layer. These fibres responded readily to circumferential stretch, as well as to blunt probing. 5. Twenty-seven fibres showed characteristics of having endings in the serosal layer. They adapted rapidly to circumferential stretch and responded to blunt probing of the serosa. Fifteen of 19 serosal fibres tested also responded to luminal chemicals. 6. Three fibres were unresponsive to all mechanical stimuli but were recruited by chemical stimuli. 7. This is the first characterization of colonic afferent fibres using an in vitro method and the first documentation of afferent fibres with their endings in the mucosa of the colon. These fibres are likely to be important in aspects of colonic sensation and reflex control.

Figure 1. Diagram of the organ bath used to record from colonic afferents

The bath had two compartments. The compartment on the left with two chambers held the colon, which was opened out and placed mucosa side uppermost over the basement chamber. The distal colon was pinned down on the right (mesenteric) side. The left side was attached to a pulley system via silk thread. Modified Krebs solution was superfused over both surfaces of the colon. Drugs were applied locally to the site of the receptive field which was isolated with a metal ring. Single fibre recordings were taken from the intermesenteric-lumbar splanchnic nerve bundle which had been passed through into the right-hand oil-filled compartment.

Figure 2. Location of afferent receptive fields and resting discharge rates of afferent fibres

A, diagram of the location of receptive fields of all afferents in the serosa, mucosa and muscle in the distal colon. Each circle represents the site, not the size of the receptive field. Shaded circles represent serosal receptive fields, filled circles represent muscular receptive fields and open circles represent mucosal receptive fields. Abbreviations: LCN, lumbar colonic nerve; IMG, inferior mesenteric ganglion; IMN-LSN, intermesenteric nerve and lumbar splanchnic nerves. B, resting discharge rate of each category of afferent fibre. Discharge was measured at the beginning of each experiment upon equilibration to experimental conditions. Mucosal afferents had significantly lower rates of resting activity than both serosal and muscular afferents (* P < 0.01 vs. serosal and P < 0.05 vs. muscular).

Figure 3. Response of a mucosal afferent to calibrated von Frey hairs and two chemical stimuli: 308 mM NaCl and undiluted ferret bile

In A-C, the integrated discharge of the fibre of interest is shown directly above the raw record of activity in the nerve strand. Three fibres were active in this strand, the discriminator templates for which are shown in D. The fibre of interest (top template) had the largest amplitude (175-200 μV). One other fibre (middle template) was subsequently investigated and included in the serosal fibre data. The last fibre (bottom template) was not investigated. A, a brief, rapidly adapting response occurred with each application of the von Frey probe. The fibre had no spontaneous activity at this stage. B, the fibre responded to the application of bile with a latency of 40 s. The response was slowly adapting and was maintained after reintroduction of the normal superfusate. C, hypertonic NaCl (308 mM) was applied to the tissue 32 min after application of bile. Basal resting discharge had increased after the application of bile upon which the response to hypertonic NaCl was superimposed. Hypertonic NaCl evoked a slowly adapting response with a latency of 10 s. This fibre was also investigated with 50 mM HCl and 100 μM capsaicin, but did not respond to these stimuli (data not shown). An illustration of the precise location of the receptive field of this fibre is shown in E. In this and subsequent figures, the calibration bar for the raw record of activity in A also applies to the other continuous raw records shown.

Figure 4. Response of a serosal fibre to circumferential stretch and blunt probing

In A and B, the integrated record of the activity of the fibre is shown directly above the raw record. Three fibres were active in this strand; the unit of interest is the one with the largest amplitude (140-150 μV) (C). A single action potential was all that was evoked by circumferential stretch (A), whereas a robust discharge was seen during probing (B). An illustration of the precise location of the receptive field of this fibre is shown in D.

Figure 5. Response of a serosal afferent to one mechanical and three chemical stimuli

In A-D, the integrated record of the activity of the fibre is shown directly above the raw record. Only the unit of interest was active in this strand. A, the unit responded to probing with a burst of firing that resolved when the stimulus was removed. Subsequent responses to probing were of lower intensity than the initial response. Moving the probe in and out of the bathing solution regularly produced artefacts. This can be observed where the raw record appears not to correspond with the integrated record. B, the fibre had a slowly adapting response to hypertonic NaCl (308 mM) with a latency of 22 s. The response was not sustained after washout with Krebs solution and the firing rate quickly returned to resting activity. C, the onset of the response to bile had a long latency of 52 s whereupon the firing pattern changed to short intense bursts of activity that had not been observed until this time. Washing the tissue with Krebs solution caused a prompt decrease in the intensity of the firing, but the bursting pattern was maintained for a further 60 s. D, the short latency (4 s) response to 50 mM HCl began with an intense burst of activity that was not sustained for the duration of the stimulus. Rather, a series of rapid, discrete bursts of firing was initiated that was sustained for the remaining 8 min of the recording period (not shown). E shows the template for this unit. F, an illustration of the location of the receptive field.

Figure 6. Response of a muscular afferent to blunt probing and circumferential stretch

In A and B, the integrated record of the activity of the fibre is shown directly above the raw record. Only the unit of interest was active in this strand. This unit had a low level of spontaneous activity. It responded to probing (A) with a burst of firing that stopped abruptly when the stimulus was removed. There was a subsequent reduction in the spontaneous activity. This fibre had a slowly adapting response to circumferential stretch (B). The degree of distortion was gradually increased throughout the stimulus, resulting in a gradual increase in the rate of firing. When the stretch was released, a brief reduction in activity below resting level occurred. The shape of the action potential is shown in C. The location of the receptive field is illustrated in D.