{alpha}7-nAChR-mediated suppression of hyperexcitability of colonic dorsal root ganglia neurons in experimental colitis

Abstract

Controlled clinical trials of nicotine transdermal patch for treatment of ulcerative colitis have been shown to improve histological and global clinical scores of colitis. Here we report that nicotine (1 microM) suppresses in vitro hyperexcitability of colonic dorsal root ganglia (DRG) (L(1)-L(2)) neurons in the dextran sodium sulfate (DSS)-induced mouse model of acute colonic inflammation. Nicotine gradually reduced regenerative multiple-spike action potentials in colitis mice to a single action potential. Nicotine's effect on hyperexcitability of inflamed neurons was blocked in the presence of an alpha(7)-nicotinic acetylcholine receptor (nAChR) antagonist, methyllicaconitine, while choline, the alpha(7)-nAChR agonist, induced a similar effect to that of nicotine. Consistent with these findings, nicotine failed to suppress hyperexcitability in colonic DRG neurons from DSS-treated alpha(7) knockout mice. Furthermore, colonic DRG neurons from DSS-treated alpha(7) knockout mice were characterized by lower rheobase (10 +/- 5 vs. 77 +/- 13 pA, respectively) and current threshold (28 +/- 4 vs. 103 +/- 8 pA, respectively) levels than DSS-treated C57BL/J6 mice. An interesting observation of this study is that 8 of 12 colonic DRG (L(1)-L(2)) neurons from control alpha(7) knockout mice exhibited multiple-spike action potential firing while no wild-type neurons did. Overall, our findings suggest that nicotine at low 1 microM concentration suppresses in vitro hyperexcitability of inflamed colonic DRG neurons in a mouse model of acute colonic inflammation via activation of alpha(7)-nAChRs.

Fig. 1.

Effect of dextran sodium sulfate (DSS) treatment on excitability of colonic sensory neurons in C57BL/J6 mice. A and B: representative traces show the effect of colitis on rheobase and firing pattern in control (CTL) (A) and inflamed (B) neurons. C: inflamed neurons showed significantly lower rheobase than control neurons and fired multiple action potentials with a further increase of the amplitude of injected current (current threshold). *Significant difference from control neurons (P = 0.004). The duration of current injections was 500 ms in A–C. D: resting membrane potentials were similar in control and inflamed neurons.

Fig. 2.

Nicotine in vitro suppresses action potential firing in inflamed colonic sensory neurons from C57BL/J6 mice. A and B: representative recordings of action potentials generated by a 500-ms current injection in the absence and presence of nicotine (1 μM) taken in 1-min intervals in colonic sensory neurons from DSS-treated (A) and control (B) C57BL/J6 mice. C: time course of the suppressive effect of nicotine on action potential discharge in neurons from DSS-treated and control C57BL/J6 mice. D: membrane potential of neurons from DSS-treated but not from control mice was shifted toward more positive values upon nicotine incubation. E: current activation in a representative inflamed neuron by prolonged application of 1 μM nicotine. Holding potential was −60 mV.

Fig. 3.

Effect of DSS-induced colonic inflammation on α₇-nicotinic acetylcholine receptor (α₇-nAChR) functional activity in colonic neurons from C57BL/J6 mice. A: a variety of nAChR-mediated currents in control colonic dorsal root ganglia (DRG) neurons isolated from a C57BL/J6 mouse. An example of ACh (1 mM)-evoked current that was suppressed by methyllicaconitine (MLA), suggesting the α₇-nAChR activation, is shown in top. An example of ACh-evoked current that was suppressed by dihydro-β-erythroidine (DHβE) but was insensitive to MLA, suggesting α₄β₂-nAChR activation, is shown in middle. The third type of current was insensitive to both DHβE and MLA, suggesting that the current was not due either to α₄β₂- or to α₇-nAChR activation, is shown in bottom. All three types of current were equally distributed among the neurons that responded to ACh. B: MLA-sensitive currents predominated in inflamed neurons, suggesting that the currents were due to α₇-nAChR activation. Examples are shown for two individual inflamed neurons from DSS-treated C57BL/J6 mouse. C: an increase in a relative number of neurons responding to ACh and sensitive to MLA in inflamed mice. Holding potential was −80 mV.

Fig. 4.

Assessment of α₇-mRNA expression in control and inflamed (INF) colonic DRG (L₁–L₂) neurons. RNA agarose gel shows single bands for α₇ and internal control, 18S.

Fig. 5.

Pharmacological evidence of essential role of α₇-nAChRs in the mechanism of nicotine-induced suppression of action potential firing in inflamed colonic neurons. A: nicotine fails to suppress multiple-spike action potential firing in inflamed C57BL/J6 neurons in the presence of the α₇ competitive antagonist MLA. B: α₇ agonist choline suppresses hyperexcitability of inflamed colonic sensory neurons. The duration of current injections was 500 ms.

Fig. 6.

Nicotine-induced suppression of action potential firing failed to occur in inflamed colonic neurons from DSS-treated α₇ knockout mice. A: nicotine fails to suppress action potential firing in the representative inflamed colonic neuron in the absence of α₇-nAChR. B: the average no. of spikes evaluated at the current threshold in individual inflamed neurons from α₇ knockout mice remains unchanged upon application of nicotine. C: membrane potential is similar in neurons from control and DSS-treated α₇ knockout mice and is not altered upon incubation with nicotine in inflamed neurons. D: rheobase and threshold levels in control and DSS-treated α₇ knockout mice. The duration of current injections was 500 ms in A, B, and D.