The origin of segmentation motor activity in the intestine

Abstract

The segmentation motor activity of the gut that facilitates absorption of nutrients was first described in the late 19th century, but the fundamental mechanisms underlying it remain poorly understood. The dominant theory suggests alternate excitation and inhibition from the enteric nervous system. Here we demonstrate that typical segmentation can occur after total nerve blockade. The segmentation motor pattern emerges when the amplitude of the dominant pacemaker, the slow wave generated by interstitial cells of Cajal associated with the myenteric plexus (ICC-MP), is modulated by the phase of induced lower frequency rhythmic transient depolarizations, generated by ICC associated with the deep muscular plexus (ICC-DMP), resulting in a waxing and waning of the amplitude of the slow wave and a rhythmic checkered pattern of segmentation motor activity. Phase-amplitude modulation of the slow waves points to an underlying system of coupled nonlinear oscillators originating in the networks of ICC.

Figure 1. Segmentation activity in the intestine in the presence of nerve blockade

a. Spatio-temporal map of in vivo mouse small intestine segmentation contractions. We describe it here as a checkered pattern, very similar to the original description of Cannon (c). The top shows normal propulsion in the stomach, White is narrowing of the lumen caused by circular muscle contraction in this and subsequent figures. Scale bars: 0.5 cm, 5s. Rectangle identifies the section enlarged in (d). b. Spatio-temporal map of in-vivo mouse small intestine peristaltic activity. Typical propulsive contractions are seen at the slow wave frequency of 32 cpm. The top shows stomach peristaltic contractions. Scale bars: 0.5 cm, 5s. c. The segmentation motor patterns as drawn by Cannon in 1902 (figure modified from). Reproduced with permission. d. A section cut out of panel (a) and turned 90 degrees for direct comparison with the drawing from Canon (c). Scale bars: 1cm, 1s. e. Spatio-temporal map of segmentation motor activity in the isolated whole mouse intestine in the presence of TTX (1 μM). Scale bars: 1cm, 1s. f. Still image from the intestine from which (e) was derived. Scale bar: 1 cm g. Spatio-temporal map of segmentation motor activity from the same experiment as panel (e) but displayed for a longer period of time. Scale bars: 1cm, 10s.

Figure 2. Association between segmentation and waxing and waning patterns

a. Spatiotemporal map shows normal propulsive motor pattern of the rat intestine before addition of decanoic acid. Scale bars: 0.5cm, 5 s. b. Amplitude profile (degree of narrowing of the lumen) taken at one point of the intestine showing regular amplitude contractions over time. Scale bars: 0.2 cm, 4 s. c. Spatiotemporal map of segmentation motor activity evoked by addition of 1 mM decanoic acid. Scale bars: 0.5 cm, 5 s. d. Amplitude profile from (b) showing waxing and waning. Scale bars: 0.2 cm, 4 s. e. Still image related to (c) showing multiple contractions of the intestine dividing it into segments in the presence of decanoic acid. Scale bar: 1 cm. f. Extracellular electrical recording, obtained at the same time as activity shown in (a). The amplitude of the slow wave is relatively constant. Scale bars: 1 mV, 10 s. g. Extracellular electrical activity obtained at the same time as activity shown in c shows a waxing and waning pattern during segmentation activity. Scale bars: 1 mV, 10 s. h. Simultaneously measured intraluminal pressure shows waxing and waning in amplitude during segmental activity shown in (c). Scale bars: 0.5 cm H₂O, 10 s.

Figure 3. Continuous wavelet transform analysis of slow wave activity and segmentation

a. Intracellular electrical activity of circular muscle in the presence decanoic acid (1 mM). MP = membrane potential b. Time-frequency contour plot of mean power (Continuous wavelet transform analysis) shows a high frequency component (~ 32 cycles/min) with a low frequency component (~3 cycles/min) in source signal a. Red and blue indicate a power increase or decrease, respectively. c. Fourier transform analysis of signal a shows complete frequency distribution of source signal (blue) and filtered low frequency signal (red). d. Signal filtering shows the presence of a low oscillatory activity in the source signal (a). Note that high frequency power in (b) is time-locked to the trough of the filtered low frequency signal in (d). MP = membrane potential. e. Diameter changes over time obtained from the spatiotemporal map (amplitude profile) in the presence of decanoic acid at one point along the intestine. f. Time-frequency contour plot of mean power of source signal (e) (continuous wavelet transform analysis) shows the high frequency (~ 38 cpm) signal amplitude with significant low frequency signals throughout (~5 cpm). Red and blue indicate a power increase or decrease, respectively. g. Fourier transform analysis of signal (e) shows complete frequency distribution of source signals (blue) and filtered low frequency signals (red). h. Significant low frequency signal shows after filtering source signal (e). Note that high frequency power in (f) is time-locked to the trough of the filtered low frequency signal in (h). The low frequency is not stationary.

Figure 4. ICC in the small intestine harbour high and low frequency activities

Live tissue c-kit staining (red) of ICC-MP (a), the layer in between ICC-MP and ICC-DMP (b) and ICC-DMP (c) using fluorescence microscopy. This is the same tissue, focussed at different depths. ICC-MP are identified by their multipolar shape, while ICC-DMP are elongated cells running parallel to circular muscle cells. d. The Fluo-4 calcium signal was recorded in the presence of decanoic acid (1mM). ICC-MP show spontaneous calcium oscillations at 30 cpm. e. From the three cells indicated by arrows in (d), the intensity (F1/F0) vs. time is shown. f. ICC-DMP show calcium oscillations at 4 cpm in the presence of decanoic acid (1mM). g. From the three cells indicated by arrows in (a), the intensity (F1/F0) vs. time is shown. Scale bars: a,b,c,d,f = 20 μm; e,g = 10s

Figure 5. Assessment of modulatory amplitude-phase coupling

a. Waxing and waning of electrical slow wave activity in the presence of decanoic acid (1 mM); 2 min recording. MP = membrane potential. b. Time-frequency contour plot of mean power (continuous wavelet transform analysis) of (a) shows high (~ 25 cpm) and low (~3cpm) frequency components. c. Modulation index of the phase-amplitude coupling (see methods). Modulation of the higher frequency (~25 cpm) component’s amplitude by the phase of a lower frequency (~3 cpm) component is measured, where higher values (towards red) indicate greater modulation. Areas within black lines indicate significance with an alpha value of 0.05 using surrogate methods. d. Intracellular electrical activity of circular muscle in the absence of decanoic acid. MP = membrane potential. e. Time-frequency contour plot of mean power shows the high frequency (~ 30 cycles/min) signal without significant low frequency signals throughout. f. No modulation (see methods) is apparent since no low frequency component is present in d. g. Waxing and waning pattern of intestine diameter recorded at one point along the small intestine during segmentation activity (2 min recording) h. Time-frequency contour plot of mean power of g. i. Modulation index carried out as in c. Highly significant modulation of the higher frequency (38 cpm) component’s amplitude by the phase of a lower frequency (~4 cpm) is seen. j. Constant amplitude intestine diameter recorded at one point along the small intestine during propulsion activity (2 min recording) k. Time-frequency contour plot of mean power shows the high frequency (~ 38 cpm) signal amplitude without significant low frequency signals throughout. l. No significant modulation is seen due to lack of significant low frequency components. m. To further demonstrate the nature of the cross frequency coupling in the segmentation waxing and waning pattern of (g), part of (g) depicted here (0.9 to 1.7 min) was band pass filtered between 0.5–5 cycles/min to obtain the low frequency component as shown. n. The higher frequency component from the signal in (m) was obtained by band pass filtering from 30–50 cycles/min and was superimposed on the lower frequency signal. A clear relationship is seen between the phase of the lower frequency and the amplitude of the higher frequency.

Figure 6. Reconstitution of the waxing and waning patterns

Reconstitution of the waxing and waning patterns of electivity activity and the checkered segmentation pattern of motor activity. a. Intracellular electrical activity in the presence of decanoic acid showing the typical waxing and waning pattern. b. Low frequency component of (a) obtained by band-pass filtering processing. c. Typical intracellular electrical activity recorded before the addition of decanoic acid. d. When the phase of the low frequency component (b) was coupled to the amplitude of the high frequency component (c) using modulated amplitude-phase coupling the typical waxing and waning pattern emerged similar to the recording in (a). e. An example of a 3.8 cm section of the intestine exhibiting a segmentation motor pattern for 30 s in the presence of lidocaine (0.5 mM). Scale bars: 0.25 cm, 2s. f. At each measured point along the intestine, the amplitude profile (degree of lightness) was taken and filtered to obtain the low frequency component. The inset shows the low frequency components of 5 consecutive positions along the intestine in between the red lines. g. Propulsive motor patterns before addition of lidocaine, the white bands representing the contractile state are uninterrupted. Scale bars: 0.25 cm, 2s. h. Here the reconstituted image is shown where each point in space is constructed using the amplitude profiles of the propulsive motor pattern (c) as the high frequency amplitude signal source and the corresponding filtered low frequency component (b) as the low frequency phase signal source to execute amplitude-phase coupling. The result is a checkered motor pattern that is similar to the recording in (a). Scale bars: 0.25 cm, 2s.

Figure 7. Phase-amplitude coupling leads to a waxing and waning slow wave pattern

Schematic using immunofluorescent images and electrical recordings to show the origin of the electrical pacemaker activities and their interaction in the circular smooth muscle layer.