Gastrointestinal Disease Research Unit (GIDRU), Queen's University, Kingston, Ontario, K7L2V7, Canada.
Department of Molecular and Cellular Biology, University of Guelph, Ontario, N1G2W1, Canada.
J Physiol. 2020 Jun;598(11):2137-2151. doi: 10.1113/JP279075. Epub 2020 Apr 6.
The vagus nerve has been implicated in mediating behavioural effects of the gut microbiota on the central nervous system. This study examined whether the secretory products of commensal gut bacteria can modulate the excitability of vagal afferent neurons with cell bodies in nodose ganglia. Cysteine proteases from commensal bacteria increased the excitability of vagal afferent neurons via activation of protease-activated receptor 2 and modulation of the voltage dependence of Na conductance activation. Lipopolysaccharide, a component of the cell wall of gram-negative bacteria, increased the excitability of nodose ganglia neurons via TLR4-dependent activation of nuclear factor kappa B. Our study identified potential mechanisms by which gut microbiota influences the activity of vagal afferent pathways, which may in turn impact on autonomic reflexes and behaviour.
Behavioural studies have implicated vagal afferent neurons as an important component of the microbiota-gut-brain axis. However, the mechanisms underlying the ability of the gut microbiota to affect vagal afferent pathways are unclear. We examined the effect of supernatant from a community of 33 commensal gastrointestinal bacterial derived from a healthy human donor (microbial ecosystem therapeutics; MET-1) on the excitability of mouse vagal afferent neurons. Perforated patch clamp electrophysiology was used to measure the excitability of dissociated nodose ganglion (NG) neurons. NG neuronal excitability was assayed by measuring the amount of current required to elicit an action potential, the rheobase. MET-1 supernatant increased the excitability of NG neurons by hyperpolarizing the voltage dependence of activation of Na conductance. The increase in excitability elicited by MET-1 supernatant was blocked by the cysteine protease inhibitor E-64 (30 nm). The protease activated receptor-2 (PAR ) antagonist (GB 83, 10 μm) also blocked the effect of MET-1 supernatant on NG neurons. Supernatant from Lactobacillus paracasei 6MRS, a component of MET-1, recapitulated the effect of MET-1 supernatant on NG neurons. Lastly, we compared the effects of MET-1 supernatant and lipopolysaccharide (LPS) from Escherichia coli 05:B5 on NG neuron excitability. LPS increased the excitability of NG neurons in a toll-like receptor 4 (TLR )-dependent and PAR -independent manner, whereas the excitatory effects of MET-1 supernatant were independent of TLR activation. Together, our findings suggest that cysteine proteases from commensal bacteria increase the excitability of vagal afferent neurons by the activation of PAR .
迷走神经在介导肠道微生物群对中枢神经系统的行为影响方面起作用。本研究旨在探讨共生肠道细菌的分泌物是否可以通过调制位于结状神经节中的迷走传入神经元的兴奋性来调节迷走传入神经元的兴奋性。共生细菌的半胱氨酸蛋白酶通过激活蛋白酶激活受体 2 和调制钠电导激活的电压依赖性来增加迷走传入神经元的兴奋性。革兰氏阴性菌细胞壁的组成部分脂多糖通过 TLR4 依赖性核因子 kappa B 的激活增加结状神经节神经元的兴奋性。我们的研究确定了肠道微生物群影响迷走传入途径活性的潜在机制，这可能反过来影响自主反射和行为。
行为研究表明迷走传入神经元是微生物群-肠道-大脑轴的一个重要组成部分。然而，肠道微生物群影响迷走传入途径的能力的潜在机制尚不清楚。我们研究了源自健康人类供体的 33 种共生胃肠道细菌的群落（微生物生态治疗；MET-1）的上清液对小鼠迷走传入神经元兴奋性的影响。穿孔膜片钳电生理学用于测量分离的结状神经节（NG）神经元的兴奋性。通过测量引起动作电位所需的电流来测定 NG 神经元的兴奋性，即 rheobase。MET-1 上清液通过超极化钠电导激活的电压依赖性来增加 NG 神经元的兴奋性。MET-1 上清液引起的兴奋性增加被半胱氨酸蛋白酶抑制剂 E-64（30nm）阻断。蛋白酶激活受体 2（PAR ）拮抗剂（GB83，10μm）也阻断了 MET-1 上清液对 NG 神经元的作用。MET-1 的组成部分副干酪乳杆菌 6MRS 的上清液再现了 MET-1 上清液对 NG 神经元的作用。最后，我们比较了 MET-1 上清液和大肠杆菌 05:B5 的脂多糖（LPS）对 NG 神经元兴奋性的影响。LPS 以 Toll 样受体 4（TLR ）依赖性和 PAR 非依赖性方式增加 NG 神经元的兴奋性，而 MET-1 上清液的兴奋性效应与 TLR 激活无关。总之，我们的研究结果表明，共生细菌的半胱氨酸蛋白酶通过激活 PAR 来增加迷走传入神经元的兴奋性。
