Key pointsr 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.r 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. r 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.r 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.Abstract 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 2 ) antagonist (GB 83, 10 μM) also blocked the effect of MET-1 supernatant on NG neurons.

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Background The gut‐ microbiota‐brain axis has received increasing attention recently due to evidence that colonic microbes can affect brain function and behavior. Recent studies have demonstrated that vagal afferent neurons may be an important conduit between the gut microbiota and the brain, as it is capable of detecting mediators released from the gut microbiota. However, it is unknown whether i) the gut microbiota from healthy human stool donors affects nodose ganglion (NG) neurophysiology or ii) gut microbial dysbiosis during inflammatory bowel disease (IBD) impacts the function of vagal afferent neurons. Hypothesis Remodeling of gut microbiota during IBD increases secretion of mediators that change the excitability of vagal afferent neurons. Methods To examine the effect of IBD patients’ fecal supernatant (FS; 1:20 dilution) on the excitability of mouse vagal afferent neurons, NG neurons from C57/Bl6 mice were collected, dissociated, and incubated overnight with FS from 5 active Crohn’s disease (CD) patients, 7 active ulcerative colitis (UC) patients, and 5 healthy volunteers (HV). Current and voltage‐clamp recordings were used to assess changes in neuronal excitability and ion channel function. Results CD and UC FS significantly increased the excitability of NG neurons through a reduction in the rheobase of 40% (CD:60 cells vs. control: 54 cells), P<0.0001, Mann‐Whitney test) and 23% (n= 50 cells vs. control: 46 cells, P= 0.0038, Mann‐Whitney test) compared to their individual vehicle control neurons, respectively. This decrease in rheobase was accompanied by a two‐fold increase in the number of action potentials elicited at twice rheobase (P <0.01, Mann‐Whitney test). However, neither resting membrane potential, nor input resistance was altered in NG neurons treated with IBD FS compared with vehicle control neurons. HV FS had no effect on NG excitability. CD and UC FS significantly reduced voltage‐gated K+ currents (P= 0.0075 and P <0.0001, two‐way ANOVA followed by Sidak's multiple comparison test, respectively), but had no effect on voltage‐gated Na+ currents. The excitatory effect of CD and UC FS of NG neurons was blocked by the cysteine protease inhibitor (E64) (30 nM), but not the serine protease inhibitor (FUT175) (10 μM). In all CD patients, pre‐incubation of E64 blocked the increase in excitability by CD patient FS (CD rheobase:40.6 ± 3.6 pA vs.CD+ E64 rheobase: 73.9 ± 4.5 pA) (P<0.0001, one‐way ANOVA followed by Tukey's multiple comparison). However, E64 was only able to block the excitatory effect of FS from 5 out of 7 UC patients FS (UC rheobase:43.8 ± 4.7 pA vs. UC+ E64 rheobase: 83.5 ± 4.3 pA) (P=0.0092, one‐way ANOVA followed by Tukey's multiple comparison). The protease‐activated receptor 2 (PAR2) antagonist GB83 (10 μM) also blocked the effect of the CD and UC patient supernatant on NG neurons (P=0.0081and P=0.0116, Kruskal‐Wallis test, respectively). Conclusion FS from active IBD patients contain mediators that can excite NG neurons. Cysteine proteases directly mediates the effect of IBD F...

Background The gut-brain axis is a bidirectional connection between the gastrointestinal tract (GI)and the central nervous system. The vagus nerve has been recognized as a principal component of this axis. Vagus nerve plays important role in maintaining homeostasis and normal GI functions, its afferent fibers can detect microbiota metabolites also. Many studies have demonstrated that the upper GI tract receives dense vagal innervation, which decreases distally throughout the tract. However, the distal colon sensory innervation of the vagus nerve remains controversial. Aims To illuminate the extent to which the vagus nerve innervates the colon, to determine whether anatomical evidence exists for double-labeled vagal afferents supplying the proximal and distal colon in the nodose ganglia. Methods C57Bl/6 mice (n=8) were injected in the proximal and distal colon with alternating solutions of retrograde tracers 1.7% Fast blue (FB) and 5% of lipophilic tracer DiI. Animals were left to recover for 10–13 days then underwent cardiac perfusion. Nodose ganglia were collected and fixed in 4% paraformaldehyde. 12 um tissue sections were then analyzed under a fluorescent microscope at 350nm and 555nm wavelength. Results In total, 27% of nodose cell bodies were labeled from the entire colon. Following proximal DiI injections, the percentage of labeled cell bodies in the nodose ganglia were 24.3± 3.9%. However, we observed a lower percentage of labeled neurons from the distal colon, with 9.3± 1.4% after DiI injections. FB labelling from the distal colon was three times less than that observed for DiI. Within the nodose ganglia, 40% of all distally labelled neurons were labelled with both tracers. Conclusions These findings indicate that in mice, both distal and proximal colon receives visceral sensory innervation from the vagus nerve. Thus, providing evidence for a sensory anatomical connection of the vagus nerve between these two parts of the colon. Funding Agencies CCC, CIHR