Background The role of intestinal microbiota in the development and function of host physiology is of high interest, especially with respect to the nervous system. While strong evidence has accrued ...that intestinal bacteria alter host nervous system function, mechanisms by which this occurs have remained elusive. For this reason, we have carried out experiments examining the electrophysiological properties of neurons in the myenteric plexus of the enteric nervous system (ENS) in germ‐free (GF) mice compared with specific pathogen‐free (SPF) control mice and adult germ‐free mice that have been conventionalized (CONV‐GF) with intestinal bacteria.
Methods Segments of jejunum from 8 to 12 week old GF, SPF, and CONV‐GF mice were dissected to expose the myenteric plexus. Intracellular recordings in current‐clamp mode were made by impaling cells with sharp microelectrodes. Action potential (AP) shapes, firing thresholds, the number of APs fired at 2× threshold, and passive membrane characteristics were measured.
Key Results In GF mice, excitability was decreased in myenteric afterhyperpolarization (AH) neurons as measured by a lower resting membrane potential and by the number of APs generated at 2× threshold. The post AP slow afterhyperpolarization (sAHP) was prolonged for GF compared with SPF and CONV‐GF animals. Passive membrane characteristics were also altered in GF mice by a decrease in input resistance.
Conclusions & Inferences Here, we report the novel finding that commensal intestinal microbiota are necessary for normal excitability of gut sensory neurons and thus provide a potential mechanism for the transfer of information between the microbiota and nervous system.
Background
The microbiome is essential for normal myenteric intrinsic primary afferent neuron (IPAN) excitability. These neurons control gut motility and modulate gut–brain signaling by exciting ...extrinsic afferent fibers innervating the enteric nervous system via an IPAN to extrinsic fiber sensory synapse. We investigated effects of germ‐free (GF) status and conventionalization on extrinsic sensory fiber discharge in the mesenteric nerve bundle and IPAN electrophysiology, and compared these findings with those from specific pathogen‐free (SPF) mice. As we have previously shown that the IPAN calcium‐dependent slow afterhyperpolarization (sAHP) is enhanced in GF mice, we also examined the expression of the calcium‐binding protein calbindin in these neurons in these different animal groups.
Methods
IPAN sAHP and mesenteric nerve multiunit discharge were recorded using ex vivo jejunal gut segments from SPF, GF, or conventionalized (CONV) mice. IPANs were excited by adding 5 μM TRAM‐34 to the serosal superfusate. We probed for calbindin expression using immunohistochemical techniques.
Key Results
SPF mice had a 21% increase in mesenteric nerve multiunit firing rate and CONV mice a 41% increase when IPANs were excited by TRAM‐34. For GF mice, this increase was barely detectable (2%). TRAM‐34 changed sAHP area under the curve by −77 for SPF, +3 for GF, or −54% for CONV animals. Calbindin‐immunopositive neurons per myenteric ganglion were 36% in SPF, 24% in GF, and 52% in CONV animals.
Conclusions & Inferences
The intact microbiome is essential for normal intrinsic and extrinsic nerve function and gut–brain signaling.
This study was conducted to assess both intrinsic and extrinsic enteric nerve function in the presence and absence of colonizing gut microbiota. Myenteric neuron slow afterhyperpolarization and mesenteric nerve multiunit discharge were recorded from the jejunum of SPF, GF, and CONV mice and compared after addition of TRAM‐34, and immunohistochemistry experiments were conducted to determine calbindin expression in same. Here, we show that an intact microbiome is essential for normal gut–brain signaling and gut calcium‐binding protein expression.
Key points
Certain probiotic bacteria have been shown to reduce distension‐dependent gut pain, but the mechanisms involved remain obscure.
Live luminal Lactobacillus reuteri (DSM 17938) and its ...conditioned medium dose dependently reduced jejunal spinal nerve firing evoked by distension or capsaicin, and 80% of this response was blocked by a specific TRPV1 channel antagonist or in TRPV1 knockout mice.
The specificity of DSM action on TRPV1 was further confirmed by its inhibition of capsaicin‐induced intracellular calcium increases in dorsal root ganglion neurons. Another lactobacillus with ability to reduce gut pain did not modify this response.
Prior feeding of rats with DSM inhibited the bradycardia induced by painful gastric distension.
These results offer a system for the screening of new and improved candidate bacteria that may be useful as novel therapeutic adjuncts in gut pain.
Certain bacteria exert visceral antinociceptive activity, but the mechanisms involved are not determined. Lactobacillus reuteri DSM 17938 was examined since it may be antinociceptive in children. Since transient receptor potential vanilloid 1 (TRPV1) channel activity may mediate nociceptive signals, we hypothesized that TRPV1 current is inhibited by DSM. We tested this by examining the effect of DSM on the firing frequency of spinal nerve fibres in murine jejunal mesenteric nerve bundles following serosal application of capsaicin. We also measured the effects of DSM on capsaicin‐evoked increase in intracellular Ca2+ or ionic current in dorsal root ganglion (DRG) neurons. Furthermore, we tested the in vivo antinociceptive effects of oral DSM on gastric distension in rats. Live DSM reduced the response of capsaicin‐ and distension‐evoked firing of spinal nerve action potentials (238 ± 27.5% vs. 129 ± 17%). DSM also reduced the capsaicin‐evoked TRPV1 ionic current in DRG neuronal primary culture from 83 ± 11% to 41 ± 8% of the initial response to capsaicin only. Another lactobacillus (Lactobacillus rhamnosus JB‐1) with known visceral anti‐nociceptive activity did not have these effects. DSM also inhibited capsaicin‐evoked Ca2+ increase in DRG neurons; an increase in Ca2+ fluorescence intensity ratio of 2.36 ± 0.31 evoked by capsaicin was reduced to 1.25 ± 0.04. DSM releasable products (conditioned medium) mimicked DSM inhibition of capsaicin‐evoked excitability. The TRPV1 antagonist 6‐iodonordihydrocapsaicin or the use of TRPV1 knock‐out mice revealed that TRPV1 channels mediate about 80% of the inhibitory effect of DSM on mesenteric nerve response to high intensity gut distension. Finally, feeding with DSM inhibited perception in rats of painful gastric distension. Our results identify a specific target channel for a probiotic with potential therapeutic properties.
Background
Environmental stress affects the gut with dysmotility being a common consequence. Although a variety of microbes or molecules may prevent the dysmotility, none reverse the dysmotility.
...Methods
We have used a 1 hour restraint stress mouse model to test for treatment effects of the neuroactive microbe, L. rhamnosus JB‐1™. Motility of fluid‐filled ex vivo gut segments in a perfusion organ bath was recorded by video and migrating motor complexes measured using spatiotemporal maps of diameter changes.
Key Results
Stress reduced jejunal and increased colonic propagating contractile cluster velocities and frequencies, while increasing contraction amplitudes for both. Luminal application of 10E8 cfu/mL JB‐1 restored motor complex variables to unstressed levels within minutes of application. L. salivarius or Na.acetate had no treatment effects, while Na.butyrate partially reversed stress effects on colonic frequency and amplitude. Na.propionate reversed the stress effects for jejunum and colon except on jejunal amplitude.
Conclusions & Inferences
Our findings demonstrate, for the first time, a potential for certain beneficial microbes as treatment of stress‐induced intestinal dysmotility and that the mechanism for restoration of function occurs within the intestine via a rapid drug‐like action on the enteric nervous system.
Environmental stress promotes gut dysmotility characterized by increased colonic and reduced small intestine propagating contractile cluster activity. We measured propulsive motility in mouse jejunum and colon ex vivo after restraint stress. Luminal Lactobacillus rhamnosus JB‐1 restored stress‐induced motility changes within minutes of application. Beneficial microbes may be useful clinically to treat stress‐induced gut dysmotility via rapid drug‐like actions on the enteric nervous system.
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