Squalamine reverses age-associated changes of firing patterns of myenteric sensory neurons and vagal fibres.

Vagus nerve signaling is a key component of the gut-brain axis and regulates diverse physiological processes that decline with age. Gut to brain vagus firing patterns are regulated by myenteric intrinsic primary afferent neuron (IPAN) to vagus neurotransmission. It remains unclear how IPANs or the afferent vagus age functionally. Here we identified a distinct ageing code in gut to brain neurotransmission defined by consistent differences in firing rates, burst durations, interburst and intraburst firing intervals of IPANs and the vagus, when comparing young and aged neurons. The aminosterol squalamine changed aged neurons firing patterns to a young phenotype. In contrast to young neurons, sertraline failed to increase firing rates in the aged vagus whereas squalamine was effective. These results may have implications for improved treatments involving pharmacological and electrical stimulation of the vagus for age-related mood and other disorders. For example, oral squalamine might be substituted for or added to sertraline for the aged.

Bacterial membrane vesicles and phages in blood after consumption of lacticaseibacillus rhamnosus JB-1.

Gut microbiota have myriad roles in host physiology, development, and immunity. Though confined to the intestinal lumen by the epithelia, microbes influence distal systems via poorly characterized mechanisms. Recent work has considered the role of extracellular vesicles in interspecies communication, but whether they are involved in systemic microbe-host interaction is unclear. Here, we show that distinctive nanoparticles can be isolated from mouse blood within 2.5 h of consuming Lacticaseibacillus rhamnosus JB-1. In contrast to blood nanoparticles from saline-fed mice, they reproduced lipoteichoic acid-mediated immune functions of the original bacteria, including activation of TLR2 and increased IL-10 expression by dendritic cells. Like the fed bacteria, they also reduced IL-8 induced by TNF in an intestinal epithelial cell line. Though enriched for host neuronal proteins, these isolated nanoparticles also contained proteins and viral (phage) DNA of fed bacterial origin. Our data strongly suggest that oral consumption of live bacteria rapidly leads to circulation of their membrane vesicles and phages and demonstrate a nanoparticulate pathway whereby beneficial bacteria and probiotics may systemically affect their hosts.

Identification of SSRI-evoked antidepressant sensory signals by decoding vagus nerve activity.

The vagus nerve relays mood-altering signals originating in the gut lumen to the brain. In mice, an intact vagus is required to mediate the behavioural effects of both intraluminally applied selective serotonin reuptake inhibitors and a strain of Lactobacillus with antidepressant-like activity. Similarly, the prodepressant effect of lipopolysaccharide is vagus nerve dependent. Single vagal fibres are broadly tuned to respond by excitation to both anti- and prodepressant agents, but it remains unclear how neural responses encode behaviour-specific information. Here we demonstrate using ex vivo experiments that for single vagal fibres within the mesenteric neurovascular bundle supplying the mouse small intestine, a unique neural firing pattern code is common to both chemical and bacterial vagus-dependent antidepressant luminal stimuli. This code is qualitatively and statistically discernible from that evoked by lipopolysaccharide, a non-vagus-dependent antidepressant or control non-antidepressant Lactobacillus strain and are not affected by sex status. We found that all vagus dependent antidepressants evoked a decrease in mean spike interval, increase in spike burst duration, decrease in gap duration between bursts and increase in intra-burst spike intervals. Our results offer a novel neuronal electrical perspective as one explanation for mechanisms of action of gut-derived vagal dependent antidepressants. We expect that our ex vivo individual vagal fibre recording model will improve the design and operation of new, extant electroceutical vagal stimulation devices currently used to treat major depression. Furthermore, use of this vagal antidepressant code should provide a valuable screening tool for novel potential oral antidepressant candidates in preclinical animal models.

Membrane vesicles of Lacticaseibacillus rhamnosus JB-1 contain immunomodulatory lipoteichoic acid and are endocytosed by intestinal epithelial cells.

Intestinal bacteria have diverse and complex influence on their host. Evidence is accumulating that this may be mediated in part by bacterial extracellular membrane vesicles (MV), nanometer-sized particles important for intercellular communication. Little is known about the composition of MV from gram-positive beneficial bacteria nor how they interact with intestinal epithelial cells (IEC). Here we demonstrate that MV from Lacticaseibacillus rhamnosus JB-1 are endocytosed in a likely clathrin-dependent manner by both mouse and human IEC in vitro and by mouse IEC in vivo. We further show that JB-1 MV contain lipoteichoic acid (LTA) that activates Toll-like receptor 2 (TLR2) and induces immunoregulatory interleukin-10 expression by dendritic cells in an internalization-dependent manner. By contrast, neither LTA nor TLR2 appear to be required for JB-1 MV endocytosis by IEC. These results demonstrate a novel mechanism by which bacterial MV can influence host physiology and suggest one potential route for beneficial influence of certain bacteria and probiotics.

Squalamine Restores the Function of the Enteric Nervous System in Mouse Models of Parkinson's Disease.

BACKGROUND: Parkinson's disease (PD) is a progressive neurodegenerative disorder thought to be caused by accumulation of α-synuclein (α-syn) within the brain, autonomic nerves, and the enteric nervous system (ENS). Involvement of the ENS in PD often precedes the onset of the classic motor signs of PD by many years at a time when severe constipation represents a major morbidity. Studies conducted in vitro and in vivo, have shown that squalamine, a zwitterionic amphipathic aminosterol, originally isolated from the liver of the dogfish shark, effectively displaces membrane-bound α-syn. OBJECTIVE: Here we explore the electrophysiological effect of squalamine on the gastrointestinal (GI) tract of mouse models of PD engineered to express the highly aggregating A53T human α-syn mutant. METHODS: GI motility and in vivo response to oral squalamine in PD model mice and controls were assessed using an in vitro tissue motility protocol and via fecal pellet output. Vagal afferent response to squalamine was measured using extracellular mesenteric nerve recordings from the jejunum. Whole cell patch clamp was performed to measure response to squalamine in the myenteric plexus. RESULTS: Squalamine effectively restores disordered colonic motility in vivo and within minutes of local application to the bowel. We show that topical squalamine exposure to intrinsic primary afferent neurons (IPANs) of the ENS rapidly restores excitability. CONCLUSION: These observations may help to explain how squalamine may promote gut propulsive activity through local effects on IPANs in the ENS, and further support its possible utility in the treatment of constipation in patients with PD.

Microvesicles from Lactobacillus reuteri (DSM-17938) completely reproduce modulation of gut motility by bacteria in mice.

Microvesicles are small lipid, bilayer structures (20-400 nm in diameter) secreted by bacteria, fungi, archaea and parasites involved in inter-bacterial communication and host-pathogen interactions. Lactobacillus reuteri DSM-17938 (DSM) has been shown to have clinical efficacy in the treatment of infantile colic, diarrhea and constipation. We have shown previously that luminal administration to the mouse gut promotes reduction of jejunal motility but increases that in the colon. The production of microvesicles by DSM has been characterized, but the effect of these microvesicles on gastrointestinal motility has yet to be evaluated. To investigate a potential mechanism for the effects of DSM on the intestine, the bacteria and its products have here been tested for changes in velocity, frequency, and amplitude of contractions in intact segments of jejunum and colon excised from mice. The effect of the parent bacteria (DSM) was compared to the conditioned media in which it was grown, and the microvesicles it produced. The media used to culture the bacteria (broth) was tested as a negative control and the conditioned medium was tested after the microvesicles had been removed. DSM, conditioned medium, and the microvesicles all produced comparable effects in both the jejunum and the colon. The treatments individually decreased the velocity and frequency of propagating contractile cluster contractions in the jejunum and increased them in the colon to a similar degree. The broth control had little effect in both tissues. Removal of the microvesicles from the conditioned medium almost completely eradicated their effect on motility in both tissues. These results show that the microvesicles from DSM alone can completely reproduce the effects of the whole bacteria on gut motility. Furthermore, they suggest a new approach to the formulation of orally active bacterial therapeutics and offer a novel way to begin to identify the active bacterial components.

Colonic Motility and Jejunal Vagal Afferent Firing Rates Are Decreased in Aged Adult Male Mice and Can Be Restored by an Aminosterol.

There is a general decline in gastrointestinal function in old age including decreased intestinal motility, sensory signaling, and afferent sensitivity. There is also increased prevalence of significant constipation in aged populations. We hypothesized this may be linked to reduced colonic motility and alterations in vagal-gut-brain sensory signaling. Using in vitro preparations from young (3 months) and old (18-24 months) male CD1 mice we report functional age-related differences in colonic motility and jejunal mesenteric afferent firing. Furthermore, we tested the effect of the aminosterol squalamine on colonic motility and jejunal vagal firing rate. Old mice had significantly reduced velocity of colonic migrating motor complexes (MMC) by 27% compared to young mice (p = 0.0161). Intraluminal squalamine increased colonic MMC velocity by 31% in old mice (p = 0.0150), which also had significantly reduced mesenteric afferent single-unit firing rates from the jejunum by 51% (p < 0.0001). The jejunal vagal afferent firing rate was reduced in aged mice by 62% (p = 0.0004). While the time to peak response to squalamine was longer in old mice compared to young mice (18.82 ± 1.37 min vs. 12.95 ± 0.99 min; p = 0.0182), it significantly increased vagal afferent firing rate by 36 and 56% in young and old mice, respectively (p = 0.0006, p = 0.0013). Our results show for the first time that the jejunal vagal afferent firing rate is reduced in aged-mice. They also suggest that there is translational potential for the therapeutic use of squalamine in the treatment of age-related constipation and dysmotility.

Antibiotic Driven Changes in Gut Motility Suggest Direct Modulation of Enteric Nervous System.

Antibiotic-mediated changes to the intestinal microbiome have largely been assumed to be the basis of antibiotic-induced neurophysiological and behavioral changes. However, relatively little research has addressed whether antibiotics act directly on the host nervous system to produce these changes. We aimed to identify whether acute exposure of the gastrointestinal tract to antibiotics directly modulates neuronally dependent motility reflexes, ex vivo. Motility of colon and jejunum segments in a perfusion organ bath was recorded by video and alterations to neuronally dependent propagating contractile clusters (PCC), measured using spatiotemporal maps of diameter changes. Short latency (<10 min) changes to PCC serve as an index of putative effects on the host nervous system. Bacitracin, penicillin V, and neomycin, all produced dose-dependent alterations to the velocity, frequency, and amplitude of PCC. Most significantly, colonic PCC velocity increased by 53% [probability of superiority (PS) = 87%] with 1.42 mg/ml bacitracin, 19% (PS = 81%) with 0.91 mg/ml neomycin, and 19% (PS = 86%) with 3.88 mg/ml penicillin V. Colonic frequency increased by 16% (PS = 73%) with 1.42 mg/ml bacitracin, 21% (PS = 79%) with 0.91 mg/ml neomycin, and 34% (PS = 85%) at 3.88 mg/ml penicillin V. Conversely, colonic amplitude decreased by 41% (PS = 79%) with 1.42 mg/ml bacitracin, 30% (PS = 80%) with 0.27 mg/ml neomycin and 25% (PS = 79%) at 3.88 mg/ml penicillin V. In the jejunum, antibiotic-specific changes were identified. Taken together, our findings provide evidence that acute exposure of the gastrointestinal lumen to antibiotics modulates neuronal reflexes. Future work should acknowledge the importance of this mechanism in mediating antibiotic-driven changes on gut-brain signaling.

Low-dose penicillin in early life induces long-term changes in murine gut microbiota, brain cytokines and behavior.

There is increasing concern about potential long-term effects of antibiotics on children's health. Epidemiological studies have revealed that early-life antibiotic exposure can increase the risk of developing immune and metabolic diseases, and rodent studies have shown that administration of high doses of antibiotics has long-term effects on brain neurochemistry and behaviour. Here we investigate whether low-dose penicillin in late pregnancy and early postnatal life induces long-term effects in the offspring of mice. We find that penicillin has lasting effects in both sexes on gut microbiota, increases cytokine expression in frontal cortex, modifies blood-brain barrier integrity and alters behaviour. The antibiotic-treated mice exhibit impaired anxiety-like and social behaviours, and display aggression. Concurrent supplementation with Lactobacillus rhamnosus JB-1 prevents some of these alterations. These results warrant further studies on the potential role of early-life antibiotic use in the development of neuropsychiatric disorders, and the possible attenuation of these by beneficial bacteria.

Effects of Saccharomyces cerevisiae or boulardii yeasts on acute stress induced intestinal dysmotility.

AIM: To investigate the capacity of Saccharomyces cerevisiae (S. cerevisiae) and Saccharomyces boulardii (S. boulardii) yeasts to reverse or to treat acute stress-related intestinal dysmotility. METHODS: Adult Swiss Webster mice were stressed for 1 h in a wire-mesh restraint to induce symptoms of intestinal dysmotility and were subsequently killed by cervical dislocation. Jejunal and colon tissue were excised and placed within a tissue perfusion bath in which S. cerevisiae, S. boulardii, or their supernatants were administered into the lumen. Video recordings of contractility and gut diameter changes were converted to spatiotemporal maps and the velocity, frequency, and amplitude of propagating contractile clusters (PCC) were measured. Motility pre- and post-treatment was compared between stressed animals and unstressed controls. RESULTS: S. boulardii and S. cerevisiae helped to mediate the effects of stress on the small and large intestine. Restraint stress reduced jejunal transit velocity (mm/s) from 2.635 ± 0.316 to 1.644 ± 0.238, P < 0.001 and jejunal transit frequency (Hz) from 0.032 ± 0.008 to 0.016 ± 0.005, P < 0.001. Restraint stress increased colonic transit velocity (mm/s) from 0.864 ± 0.183 to 1.432 ± 0.329, P < 0.001 and frequency to a lesser degree. Luminal application of S. boulardii helped to restore jejunal and colonic velocity towards the unstressed controls; 1.833 ± 0.688 to 2.627 ± 0.664, P < 0.001 and 1.516 ± 0.263 to 1.036 ± 0.21, P < 0.001, respectively. S. cerevisiae also had therapeutic effects on the stressed gut, but was most apparent in the jejunum. S. cerevisiae increased PCC velocity in the stressed jejunum from 1.763 ± 0.397 to 2.017 ± 0.48, P = 0.0031 and PCC frequency from 0.016 ± 0.009 to 0.027 ± 0.007, P < 0.001. S. cerevisiae decreased colon PCC velocity from 1.647 ± 0.187 to 1.038 ± 0.222, P < 0.001. Addition of S. boulardii or S. cerevisiae supernatants also helped to restore motility to unstressed values in similar capacity. CONCLUSION: There is a potential therapeutic role for S. cerevisiae and S. boulardii yeasts and their supernatants in the treatment of acute stress-related gut dysmotility.

Magnetic resonance spectroscopy reveals oral Lactobacillus promotion of increases in brain GABA, N-acetyl aspartate and glutamate.

The gut microbiome has been shown to regulate the development and functions of the enteric and central nervous systems. Its involvement in the regulation of behavior has attracted particular attention because of its potential translational importance in clinical disorders, however little is known about the pathways involved. We previously have demonstrated that administration of Lactobacillus rhamnosus (JB-1) to healthy male BALB/c mice, promotes consistent changes in GABA-A and -B receptor sub-types in specific brain regions, accompanied by reductions in anxiety and depression-related behaviors. In the present study, using magnetic resonance spectroscopy (MRS), we quantitatively assessed two clinically validated biomarkers of brain activity and function, glutamate+glutamine (Glx) and total N-acetyl aspartate+N-acetyl aspartyl glutamic acid (tNAA), as well as GABA, the chief brain inhibitory neurotransmitter. Mice received 1×10(9) cfu of JB-1 per day for 4weeks and were subjected to MRS weekly and again 4weeks after cessation of treatment to ascertain temporal changes in these neurometabolites. Baseline concentrations for Glx, tNAA and GABA were equal to 10.4±0.3mM, 8.7±0.1mM, and 1.2±0.1mM, respectively. Delayed increases were first seen for Glx (~10%) and NAA (~37%) at 2weeks which persisted only to the end of treatment. However, Glx was still elevated 4weeks after treatment had ceased. Significantly elevated GABA (~25%) was only seen at 4weeks. These results suggest specific metabolic pathways in our pursuit of mechanisms of action of psychoactive bacteria. They also offer through application of standard clinical neurodiagnostic techniques, translational opportunities to assess biomarkers accompanying behavioral changes induced by alterations in the gut microbiome.

The TRPV1 channel in rodents is a major target for antinociceptive effect of the probiotic Lactobacillus reuteri DSM 17938.

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 Ca(2+) 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 Ca(2+) increase in DRG neurons; an increase in Ca(2+) 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.

Gut commensal microvesicles reproduce parent bacterial signals to host immune and enteric nervous systems.

Ingestion of a commensal bacteria, Lactobacillus rhamnosus JB-1, has potent immunoregulatory effects, and changes nerve-dependent colon migrating motor complexes (MMCs), enteric nerve function, and behavior. How these alterations occur is unknown. JB-1 microvesicles (MVs) are enriched for heat shock protein components such as chaperonin 60 heat-shock protein isolated from Escherichia coli (GroEL) and reproduce regulatory and neuronal effects in vitro and in vivo. Ingested labeled MVs were detected in murine Peyer's patch (PP) dendritic cells (DCs) within 18 h. After 3 d, PP and mesenteric lymph node DCs assumed a regulatory phenotype and increased functional regulatory CD4(+)25(+)Foxp3+ T cells. JB-1, MVs, and GroEL similarly induced phenotypic change in cocultured DCs via multiple pathways including C-type lectin receptors specific intercellular adhesion molecule-3 grabbing non-integrin-related 1 and Dectin-1, as well as TLR-2 and -9. JB-1 and MVs also decreased the amplitude of neuronally dependent MMCs in an ex vivo model of peristalsis. Gut epithelial, but not direct neuronal application of, MVs, replicated functional effects of JB-1 on in situ patch-clamped enteric neurons. GroEL and anti-TLR-2 were without effect in this system, suggesting the importance of epithelium neuron signaling and discrimination between pathways for bacteria-neuron and -immune communication. Together these results offer a mechanistic explanation of how Gram-positive commensals and probiotics may influence the host's immune and nervous systems.

Fucosylated but not sialylated milk oligosaccharides diminish colon motor contractions.

Human milk oligosaccharides (HMO) are being studied by different groups exploring a broad range of potential beneficial effects to the breastfed infant. Many of these effects have been attributed to a growth promotion effect on certain gut organisms such as bifidobacteria. Additionally, evidence indicates that HMO are able to directly promote positive changes in gut epithelium and immune responses under certain conditions. This study utilizes a standardized ex vivo murine colon preparation to examine the effects of sialylated, fucosylated and other HMO on gut motor contractions. Only the fucosylated molecules, 2'FL and 3'FL, decreased contractility in a concentration dependent fashion. On the basis of IC50 determinations 3'FL was greater than 2 times more effective than 2'FL. The HMO 3'SL and 6'SL, lacto-N-neotetraose (LNnT), and galactooligosaccharides (GOS) elicited no effects. Lactose was used as a negative control. Fucosylation seems to underlie this functional regulation of gut contractility by oligosaccharides, and L-fucose, while it was also capable of reducing contractility, was substantially less effective than 3'FL and 2'FL. These results suggest that specific HMO are unlikely to be having these effects via bifidogenesis, but though direct action on neuronally dependent gut migrating motor complexes is likely and fucosylation is important in providing this function, we cannot conclusively shown that this is not indirectly mediated. Furthermore they support the possibility that fucosylated sugars and fucose might be useful as therapeutic or preventative adjuncts in disorders of gut motility, and possibly also have beneficial central nervous system effects.