Synaptic facilitation and enhanced neuronal excitability in the submucosal plexus during experimental colitis in guinea-pig

Abstract

Intestinal secretion is regulated by submucosal neurones of the enteric nervous system. Inflammation of the intestines leads to aberrant secretory activity; therefore we hypothesized that the synaptic and electrical behaviours of submucosal neurones are altered during colitis. To test this hypothesis, we used intracellular microelectrode recording to compare the excitability and synaptic properties of submucosal neurones from normal and trinitrobenzene sulphonic acid (TNBS)-inflamed guinea-pig colons. Inflammation differentially affected the electrophysiological characteristics of the two functional classes of submucosal neurones. AH neurones from inflamed colons were more excitable, had shorter action potential durations and reduced afterhyperpolarizations. Stimulus-evoked fast and slow excitatory postsynaptic potentials (EPSPs) in S neurones were larger during colitis, and the incidence of spontaneous fast EPSPs was increased. In control preparations, fast EPSPs were almost completely blocked by the nicotinic receptor antagonist hexamethonium, whereas fast EPSPs in inflamed S neurones were only partially inhibited by hexamethonium. In inflamed tissues, components of the fast EPSP in S neurones were sensitive to blockade of P2(X) and 5-HT(3) receptors while these antagonists had little effect in control preparations. Control and inflamed S neurones were equally sensitive to brief application of acetylcholine, ATP and 5-HT, suggesting that synaptic facilitation was due to a presynaptic mechanism. Immunoreactivity for 5-HT in the submucosal plexus was unchanged by inflammation; this indicates that altered synaptic transmission was not due to anatomical remodelling of submucosal nerve terminals. This is the first demonstration of alterations in synaptic pharmacology in the enteric nervous system during inflammation.

Figure 1. Enhanced excitability of AH neurones from inflamed colon

Mean ± s.e.m. data are presented in A, B and D. A, the maximum number of APs during 500 ms, and the number of APs during 2 × rheobase depolarizations is increased in AH neurones from inflamed colon (black columns) versus controls (grey columns). B, the magnitude of the AHP, measured as the area under the curve of the AHP, is reduced during inflammation. C, representative recordings of intracellularly evoked APs in AH neurones from inflamed (black line) and control (grey line) colons. Note the reduction in AP duration during inflammation. D, population data for APD₅₀ changes during inflammation. Numbers of neurones for each measurement are indicated in the bars. *P < 0.01; **P < 0.001.

Figure 2

Regression analysis of action potential duration at half-amplitude (APD₅₀) versus afterhyperpolarizing potential (AHP) magnitude and neuronal excitability.

Figure 3. Evoked synaptic potentials are larger in S neurones from inflamed colons

A, mean ± s.e.m. amplitudes of fast EPSPs at −80 mV from control (grey columns) and inflamed (black columns) S neurones. B, slow EPSP amplitude is increased in AH neurones from inflamed colon. C, an example of the spontaneous electrical activity observed in submucosal S neurones. It consists of spontaneous fast EPSPs that are predominantly subthreshold but that can often reach threshold for action potential firing. D, spontaneous fast EPSPs, similar to those depicted in C, were observed significantly more frequently in S neurones from inflamed colons compared with controls. E, a spontaneous slow depolarization in an S neurone from inflamed colon. Note that the depolarization is preceeded by a burst of spontaneous fast EPSPs (inset), indicating neurotransmitter release onto the neurone immediately prior to the slow depolarization. Numbers of neurones for each measurement are indicated in the columns. *P < 0.01; **P < 0.001.

Figure 4. Altered pharmacology of evoked fast EPSPs in S neurones

A and B, raw data illustrating the difference in amplitude and hexamethonium sensitivity of fast EPSPs from control (A) and inflamed (B) colons. Evoked fast EPSPs in S neurones from inflamed colons (black columns) are larger and are less sensitive to blockade of nicotinic receptors than S neurones from control animals (grey columns). C, mean ± s.e.m. amplitude data for fast EPSPs, hexamethonium (100 μm)-sensitive components (nicotinic receptor components), PPADs (30 μm)-sensitive components (P2_X receptor components) and granisetron (1 μm)-sensitive components (5-HT₃ receptor components). Fast EPSP amplitude at −80 mV is larger in S neurones from inflamed colon. This is not due to an increase in the amplitude of nicotinic receptor mediated fast EPSPs. The amplitudes of 5-HT₃ receptor and P2_X receptor components are significantly larger during inflammation and appear to underlie the increase in evoked fast EPSP amplitude following inflammation. Numbers of neurones for each measurement are indicated in the bars. *P < 0.01; **P < 0.001.

Figure 5. Membrane potential responses to applied neurotransmitter

A, raw data illustrating the depolarization caused in an S neurone by 100 ms spritz of 1 mm acetylcholine (ACh). Note the increase in conductance during the depolarization caused by opening of non-selective cation channels. B, mean ± s.e.m. amplitude data for the depolarizations of neurones in response to 100 ms spritzes of ACh (1 mm), ATP (1 mm) or 5-HT (300 μm). No significant differences were evident between control (grey columns) and inflamed (black columns) neurones in their responses to any of the ligands. There were also no significant differences in the amplitude of any of the response to the different ligands.

Figure 6. Immunohistochemical detection of 5-HT in the submucosal plexus of control and inflamed colons

Representative confocal images of 5-HT immunoreactivity in submucosal ganglia from control (A) and inflamed (B) colons. There were no detectable differences in 5-HT distribution following colitis. The images are digital composites of 28 optical sections (control) and 31 optical sections (inflamed) taken at 1 μm thickness.