Enhanced excitability of myenteric AH neurones in the inflamed guinea-pig distal colon

Abstract

The electrical and synaptic properties of myenteric neurones in normal and inflamed guinea-pig distal colons were evaluated by intracellular microelectrode recording. Chronic inflammation was established 6 days following administration of trinitrobenzene sulfonic acid (TNBS). In S neurones, inflammation only altered synaptic inputs as the amplitude of fast excitatory postsynaptic potentials were significantly larger (31 +/- 2 mV compared to 20 +/- 1 mV) and they were more likely to receive slow excitatory synaptic input (85% compared to 55%). AH neurones displayed altered electrical properties in colitis compared to control tissues: they generated more action potentials during a maximal depolarising current pulse (7 +/- 1 compared to 1.6 +/- 0.2); they had a smaller after hyperpolarisation (9 +/- 2 mV s compared to 20 +/- 2 mV s); and they were more likely to receive fast excitatory synaptic input (74% compared to 17%), possess spontaneous activity (46% compared to 3%), and generate anodal break action potentials (58% compared to 19%). Although the resting membrane potential, input resistance and action potential characteristics were unaltered in AH neurones from inflamed tissues, they exhibited an enhanced Cs+-sensitive rectification of the current-voltage relationship. This suggests that the increase in excitability of AH neurones may involve a colitis-induced augmentation of the hyperpolarisation-activated cation current (Ih) in these cells. An increased excitability, selectively in AH neurones, suggests that the afferent limb of intrinsic motor reflexes is disrupted in the inflamed colon and this may contribute to dysmotility associated with inflammatory diseases.

Figure 1. Amplitude of fast excitatory synaptic potentials in S neurones from control or inflamed distal colon

A, respresentative traces of fEPSPs elicited by fibre tract stimulation (0.5 ms 5.4 V; open arrows) in S neurones from control and inflamed tissue. Both traces are on the same voltage, current and time scale. B, scatter plot illustrating the maximum amplitude of fEPSPs in S neurones from TNBS-treated (open circles) or control colons (grey squares). Data points are displaced horizontally so that overlapping data points can be visualised. The lines demonstrate the mean ±s.e.m. for each treatment group (^*P < 0.001 compared to control, t test; n= 37 for controls and 27 for inflamed).

Figure 2. Accomodation in AH neurones from control or inflamed distal colon

A, representative traces of a response to a 500 ms depolarising current pulse in AH neurones from control and inflamed tissue. Both traces are on the same voltage, current and time scale. B, scatter plot illustrating the maximum number of action potentials in response to a 500 ms depolarising current pulse in AH neurones from TNBS-treated (open circles) or control colons (grey squares). Data points are displaced horizontally so that overlapping data points can be visualised. The lines demonstrate the mean ±s.e.m. for each treatment group (^*P < 0.001 compared to control, t test; n= 36 for controls and 28 for inflamed).

Figure 3. Spontaneous activity in AH neurones from control or inflamed distal colon

A, representative traces of baseline recordings in AH neurones from control and inflamed tissue. Both traces are on the same voltage and time scale. B, bar graph illustrating the proportion of AH neurones from TNBS-treated (open bar) or control colons (grey bar) that demonstrated spontaneous activity (^*P < 0.001 compared to control, Fisher's exact test).

Figure 4. Anodal break action potentials in AH neurones from control or inflamed distal colon

A, representative traces of responses at the offset of a 500 ms hyperpolarising current pulse in AH neurones from control and inflamed tissue. Both traces are on the same voltage, current and time scale. B, bar graph illustrating the proportion of AH neurones from TNBS-treated (open bar) or control colons (grey bar) that demonstrated anodal break action potentials following a hyperpolarising current pulse (^*P < 0.001 compared to control, Fisher's exact test).

Figure 5. Slow afterhyperpolarisations in AH neurones from control or inflamed distal colon

A, representative traces of AHP following single action potentials from control and inflamed tissue. Traces for both cells are on the same voltage, current and time scales. The magnitude of the AHP, determined by integrating the voltage less than resting membrane potential over the time of the entire AHP, for these representative neurones were: control, 25 mV s; and inflamed, 5 mV s. B, scatter plot illustrating the magnitude of the AHP in AH neurones from TNBS-treated (open circles) or control colons (grey squares). Data points are displaced horizontally so that overlapping data points can be visualised. The lines demonstrate the mean ±s.e.m. for each treatment group (^*P < 0.001 compared to control, t test; n= 33 for controls and 26 for inflamed).

Figure 6. Fast EPSPs in AH neurones from control or inflamed distal colon

A, bar graph illustrating the proportion of AH neurones from TNBS-treated (open bar) or control colons (grey bar) that received fast excitatory synaptic input in response to focal stimulation of fibre tracts with single pulses (0.5 ms, 1–10 V) (^*P < 0.001 compared to control, Fisher's exact test). B, respresentative trace of one of the five AH neurones from inflamed preparations whose fEPSPs elicited by fibre tract stimulation (0.5 ms 3.6 V; open arrows) were blocked by the addition of 100 μm hexamethonium to the bathing solution.

Figure 7. Action potentials in AH neurones from control or inflamed distal colon

A, representative traces of antidromically elicited action potentials in AH neurones from control and inflamed tissue. Both traces are on the same voltage, current and time scale. Letters on the control voltage and derivitive traces indicate parameters that were measured and correspond to graphs shown in B−F. The magnitude of the action potentials for these representative neurones, determined by integrating the voltage greater than resting membrane potential over the time of the action potential, were: control, 129 mV ms; inflamed, 119 mV ms. B−F, scatter plots illustrating the maximum rate of depolarisation (B), maximum rate of repolarisation (C), duration at half-repolarisation (D), amplitude (E) and magnitude (F) of the action potentials in AH neurones from TNBS-treated (open circles) or control colons (grey squares). Data points are displaced horizontally so that overlapping data points can be visualised. The lines demonstrate the means ±s.e.m. for each treatment group (for all plots P > 0.05 compared to control, t test; n= 7 for controls and 14 for inflamed).

Figure 8. Contribution of a Cs⁺-sensitive hyperpolarisation-activated conductance to excitability in AH neurones from inflamed distal colon

A, representative hyperpolarising electrotonic potentials from AH neurones from control and inflamed preparations in the absence and presence of 2 mm CsCl. Note that the depolarising sag in the electrotonic potential is more pronounced in traces from the neurone in inflamed tissue than control tissue, and that the amplitudes of electrotonic potentials were larger in the presence of CsCl. B, a representative current–voltage plot of the steady state electrotonic potentials elicited by intracellular injection of current at a holding potential of −50 mV. C and D, using plots like that shown in B, changes in voltage provoked by Cs+ (ΔV_Cs) were measured as the difference between Cs⁺ and no Cs⁺ lines and plotted as a function of cell voltages from the Cs⁺ graph (V_Cs). There were no differences detected in the Cs⁺-sensitive instantaneous voltage (measured at the time point indicated by † in A; P > 0.05 two-way ANOVA for repeated measures). In the steady state voltage measurements (measured at the time point indicated by ‡ in A), there was a significant augmentation of the Cs⁺-sensitive voltage at potentials more negative than −90 mV (^*P < 0.05, two-way ANOVA for repeated measures, Bonferroni's multiple comparisons test). In inflamed tissue, application of CsCl to AH neurones reduced the occurence of anodal break action potentials (E), and increased the magnitude of the afterhyperpolarisation (F). E and F are representative examples from the same cell, held at −50 mV.