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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.

Background The fasting and stress associated with road transportation contributes to a lack of energy and a decline in the immune system of beef cattle. Therefore, it is essential for beef cattle to enhance energy reserves before transportation. Creatine pyruvate (CrPyr) is a new multifunctional nutrient that can provide both pyruvate and creatine, which are two intermediate products of energy metabolism. To investigate the effects of transport and rumen-protected (RP)-CrPyr on the blood biochemical parameters and rumen fluid characteristics of beef cattle, twenty male Simmental crossbred cattle (659 ± 16 kg) aged 18 months were randomly allocated to four groups (n = 5) using a 2 × 2 factorial arrangement with two RP-CrPyr supplemental levels (0 or 140 g/d) and two transport treatments (5 min or 12 h): T_CrPyr140, T_CrPyr0, NT_CrPyr140, and NT_CrPyr0. After feeding for 30 days, three cattle per treatment were slaughtered. Results Compared with nontransport, transport decreased the total antioxidant capacity, catalase activity, contents of IgA, interferon γ, interleukin-1β (IL-1β), and IL-6 in serum, and the amounts of total volatile fatty acids (TVFA), acetate, and butyrate in rumen (P P P P Prevotella genus abundance compared with group NT_CrPyr0 (P Prevotella abundance compared with group T_CrPyr0 (P Conclusions These results indicated that dietary supplementation with RP-CrPyr might be beneficial to alleviate transport stress by decreasing serum cortisol and LPS levels and promoting the restoration of the rumen natural flora.

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.

The gut-microbiota-brain axis is implicated in the development of behavioural disorders in mammals. As such, its potential role in disruptive feather pecking (FP) in birds cannot be ignored. Birds with a higher propensity to perform FP have distinct microbiota profiles and feed transit times compared to non-pecking counterparts. Consequently, we hypothesize that the gut microbiota is intimately linked to FP and gut motility, which presents the possibility of using probiotics to control FP behaviour. In the present study, we aim to assess the relationship between cecal motility and the probiotic Lactobacillus rhamnosus in chickens classified as peckers (P, 13 birds) and non-peckers (NP, 17 birds). We show that cecal contractions were 68% less frequent and their amplitude increased by 58% in the presence of L. rhamnosus. Furthermore, the number of FP bouts performed by P birds was positively correlated with contraction velocity and amplitude. We present the first account of gut motility measurements in birds with distinct FP phenotypes. Importantly, the present work demonstrates the clear impact of a probiotic on cecal contractions. These findings lay the foundation for identifying biological differences between P and NP birds which will support the development of FP control strategies.

Study design A rat model of multifidus muscles injury and atrophy after posterior lumbar spine surgery. Objective We determined the effect of ascorbic acid (AA) on the postoperative multifidus muscles in rat model. Summary of background data Previous studies show oxidative stress and inflammation are two main molecular mechanisms in multifidus muscle injury and atrophy after posterior lumbar surgery. AA may have a protective effect in postoperative multifidus muscles. Methods Rats were divided into sham surgery, control surgery, and surgery plus AA groups. Multifidus muscles of the control and AA groups were excised from the osseous structures. The muscles were retracted continuously for 2 hours. In the sham and AA groups, AA was administered via oral gavage daily in the first week. In each group, the oxidative stress was evaluated by measuring malondialdehyde (MDA) and Total superoxide dismutase (T-SOD). The inflammation, fat degeneration, or fibrosis of multifidus muscle were evaluated by quantitative real-time polymerase chain reaction (q-PCR), histology, or immunohistochemical analysis. Results T-SOD activity was significantly lower in the control group than that in the AA group in the first week. MDA levels were significantly higher in the AA group. Interleukin-6 and tumor necrosis factor-α in multifidus muscles also showed significant differences when treated with AA. The inflammation score on histology was significantly lower in the AA group postoperatively in the first week. In the long run, marker genes for fibrosis and fat degeneration, and fibrosis and fat degeneration scores, were significantly lower in the AA than the control group on days 14 and 28 postoperatively. Conclusion In conclusion, AA attenuated the oxidative stress and inflammation response in the postoperative multifidus muscles, and remarkable differences were observed from the histological assessment and related marker genes expression. Our results provided important insight into the anti-inflammatory and anti-oxidative effects of AA in the postoperative multifidus muscles. Level of evidence N/A.

Arsenic (As) is a highly toxic contaminant in the environment and a serious carcinogen for the human being. The toxicity of arsenic significantly threatens environmental and human health. The effective removing technology for arsenic remains challenging, and one of the reasons is due to the lack of powerful detection method in the complex environmental matrix. There is thus an urgent need to develop novel analytical methods for arsenic, preferably with the potential for the field-testing. To combat arsenic pollution and maintain a healthy environment and eco-system, many advanced analytical methods have been developed for arsenic detection in various samples. Among these strategies, biosensors hold great promise for rapid detection of arsenic, in particular, nanomaterials-based aptamer sensors have attracted significant attention due to their simplicity, high sensitivity and rapidness. In this paper, we reviewed the recent development and promising applications of aptamer sensors (aptasensors) based-on nanomaterial for arsenic detection, in particular with emphasis on the works using optical and electrochemical technologies. We also discussed the recent novel technology in aptasensors development for arsenic detection, including nucleic acid amplification for signal enhancement and device integration for the portability of arsenic sensors. We are hoping this review could inspire further researches in developing novel nanotechnologies based aptasensors for possible on-site detection of arsenic.

The vagus nerve can transmit signals to the brain resulting in a reduction in depressive behavior as evidenced by the long-term beneficial effects of electrical stimulation of the vagus in patients with intractable depression. The vagus is the major neural connection between gut and brain, and we have previously shown that ingestion of beneficial bacteria modulates behaviour and brain neurochemistry via this pathway. Given the high levels of serotonin in the gut, we considered if gut-brain signaling, and specifically the vagal pathway, might contribute to the therapeutic effect of oral selective serotonin reuptake inhibitors (SSRI). Mesenteric nerve recordings were conducted in mice after treatment with SSRI to ascertain if this class of drugs resulted in increased vagal excitability. Patch clamp recordings of enteric neurons were carried out to measure activity of primary afferent neurons in the gut in response to SSRI and to assess the importance of gut epithelium in transducing signal. The tail suspension test (TST) was used following 14d feeding of SSRI in vagotomised and surgical sham mice to measure depressive-like behaviour. Brain mRNA expression was examined via PCR and the intestinal microbiome was assessed. Mesenteric nerve recordings in BALB/c mice demonstrated that oral treatment with SSRI leads to a significant increase in vagal activity. This effect was not observed in mice treated with a representative noradrenaline-dopamine reuptake inhibitor. It is known that signals from the gut can be transmitted to the vagus via the enteric nervous system. Exposure of the gut to SSRI increased the excitability of intrinsic primary afferent neurons in the myenteric plexus, through an intestinal epithelium dependent mechanism, and alpha-diversity of gut microbiota was altered. Critically, blocking vagal signaling from gut to brain, via subdiaphragmatic vagotomy, abolished the antidepressive effects of oral SSRI treatment as determined by the tail suspension test. This work suggests that vagus nerve dependent gut-brain signaling contributes to the effects of oral SSRI and further, highlights the potential for pharmacological approaches to treatment of mood disorders that focus on vagal stimulation and may not even require therapeutic agents to enter the circulation.

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.

Methamphetamine (METH) has been recognized as an emerging organic contaminant as it was widely detected in the aquatic environment via wastewater effluent discharge. However, the ecological hazard posed by METH at environmentally relevant concentrations was remained unclear. In this study, adult medaka fish were exposed to METH at environmental levels (0.05, 0.2, 0.5, 5 μg L−1) and high level (25 and 100 μg L−1) for 90 days to investigate its effect on ecologically behavioral functions, histopathology, bioconcentration, and transgenerational toxicity. The significant increase of locomotion activity, total distance, and max velocity of adult medaka were observed at low METH levels (0.2–0.5 μg L−1), while it markedly decreased at high levels (25–100 μg L−1). This effect may increase the predation risk of the fish. The significant alteration on the relative expressions of the genes (cacna1c, oxtr, erk1, and c-fos), as well as the contents of the proteins (oxytocin (OXT) and protein kinase A (PKA)) involved in Voltage Dependent Calcium Channel (VDCC) and Mitogen-Activated Protein Kinase (MAPK) signaling channel induced by METH could partly elucidate the underlying mechanisms of the changes of the behavioral traits. METH could induce obvious minimal gliosis, neuronal loss, and necrotic in brain tissues. Additionally, the significant increase of hepatic-somatic index (HSI) of male medaka at 0.2–5 μg L−1 groups, and the decrease of female medaka at 100 μg L−1 group indicated male fish was more susceptible to METH. Nephric-somatic index (NSI) of medaka markedly declined induced by METH at 0.05–100 μg L−1. The bioconcentration factor (BCF) (0.4–5.8) in medaka fish revealed the bioconcentration potential of METH in fish. This study for the first time demonstrated METH could induced the development defects of larvae in F1 generation at environmentally relevant concentrations, thereby resulting in a significant decrease in the capacity of fish to produce offspring. Meanwhile, the RQ values (>1) of METH in river in China, USA, and Australia showed a high teratogenic risk level, suggesting the ecosystem-levels consequence posed by METH should be concerned.

Mounting evidence supports the influence of the gut microbiome on the local enteric nervous system and its effects on brain chemistry and relevant behavior. Vagal afferents are involved in some of these effects. We previously showed that ingestion of the probiotic bacterium Lactobacillus rhamnosus (JB-1) caused extensive neurochemical changes in the brain and behavior that were abrogated by prior vagotomy. Because information can be transmitted to the brain via primary afferents encoded as neuronal spike trains, our goal was to record those induced by JB-1 in vagal afferents in the mesenteric nerve bundle and thus determine the nature of the signals sent to the brain. Male Swiss Webster mice jejunal segments were cannulated ex vivo, and serosal and luminal compartments were perfused separately. Bacteria were added intraluminally. We found no evidence for translocation of labeled bacteria across the epithelium during the experiment. We recorded extracellular multi- and single-unit neuronal activity with glass suction pipettes. Within minutes of application, JB-1 increased the constitutive single- and multiunit firing rate of the mesenteric nerve bundle, but Lactobacillus salivarius (a negative control) or media alone were ineffective. JB-1 significantly augmented multiunit discharge responses to an intraluminal distension pressure of 31 hPa. Prior subdiaphragmatic vagotomy abolished all of the JB-1-evoked effects. This detailed exploration of the neuronal spike firing that encodes behavioral signaling to the brain may be useful to identify effective psychoactive bacteria and thereby offer an alternative new perspective in the field of psychiatry and comorbid conditions.

Gut commensals modulate host immune, endocrine, and metabolic functions. They also affect peripheral and central neural reflexes and function. We have previously shown that daily ingestion of Lactobacillus reuteri (LR) for 9 d inhibits the pseudoaffective cardiac response and spinal single-fiber discharge evoked by visceral distension, and decreases intestinal motility and myenteric AH cell slow afterhyperpolarization (sAHP) by inhibiting a Ca-activated K (IK(Ca)) channel. We tested whether luminal LR could acutely decrease motility in an ex vivo perfusion model of naive Balb/c jejunum. Live LR dose dependently decreased motor complex pressure wave amplitudes with 9- to 16-min onset latency and an IC(50) of 5 × 10(7) cells/ml Krebs. Heat-killed LR or another live commensal, Lactobacillus salivarius, were without effect. The IK(Ca) channel blocker TRAM-34, but neither the opener (DCEBIO) nor the hyperpolarization-activated cationic channel inhibitor ZD7288 (5 μM) (or TTX 1 μM), mimicked the LR effect on motility acutely ex vivo. We provide evidence for a rapid, strain-specific, dose-dependent action of a live Lactobacillus on small intestinal motility reflexes that recapitulates the long-term effects of LR ingestion. These observations may be useful as a first step to unraveling the pathways involved in bacteria to the nervous system communication.

Lactobacillus species ingestion can decrease autonomic responses and spinal fiber discharge to nociceptive colorectal distension (CRD), even in the absence of inflammation. The present study aimed to determine whether dorsal root ganglion (DRG) somas could be a locus where the antinociceptive probiotic may have an effect. Healthy rats were fed with Lactobacillus reuteri or vehicle control for 9 days whereupon they were anesthetized, and intermittent distal colonic CRD at 80 mmHg distension was either performed for 1 h or not. The animals were immediately euthanized and patch-clamp recordings taken after isolation and overnight culture from those DRG that projected to the distal colon. CRD decreased the threshold for action potential generation and increased the number of spikes discharged during a standard depolarizing test stimulus, and this effect was blocked by prior probiotic ingestion. The increase in excitability was paralleled by an increase in DRG capacitance, which was not altered by Lactobacillus reuteri ingestion. CRD did not increase tissue weight or myeloperoxidase activity. We suggest that the effects of CRD may have been caused by activity-dependent neurotransmission between DRG somas. CRD evoked increases in action potential upstroke speed, which suggests that it may also have led to augmentation of sodium channel conductances. Probiotic ingestion may have interfered with this hypothetical mechanism since it blocked the effect of CRD on the action potential.

We recorded from myenteric AH/Dogiel type II cells, demonstrated mechanosensitive responses, and characterized their basic properties. Recordings were obtained using the mouse longitudinal muscle myenteric plexus preparation with patch-clamp and sharp intracellular electrodes. The neurons had an action potential hump and a slow afterhyperpolarization (AHP) current. The slow AHP was carried by intermediate conductance Ca2+-dependent K+-channel currents sensitive to charybdotoxin and clotrimazole. All possessed a hyperpolarization-activated current that was blocked by extracellular cesium. They also expressed a TTX-resistant Na+ current with an onset near the resting potential. Pressing on the ganglion containing the patched neuron evoked depolarizing potentials in 17/18 cells. The potentials persisted after synaptic transmission was blocked. Volleys of presynaptic electrical stimuli evoked slow excitatory postsynaptic potentials (EPSPs) in 9/11 sensory neurons, but 0/29 cells received fast EPSP input. The slow EPSP was generated by removal of a voltage-insensitive K+ current. Patch-clamp recording with a KMeSO4-containing, but not a conventional KCl-rich, intracellular solution reproduced the single-spike slow AHPs and low input resistances seen with sharp intracellular recording. Cell-attached recording of intermediate conductance potassium channels supported the conclusion that the single-spike slow AHP is an intrinsic property of intestinal AH/sensory neurons. Unitary current recordings also suggested that the slow AHP current probably does not contribute significantly to the high resting background conductance seen in these cells. The characterization of mouse myenteric sensory neurons opens the way for the study of their roles in normal and pathological physiology.

Background and aims: Probiotic bacteria are being investigated as possible treatments for many intestinal disorders. The present study aimed to explore the effects of live, heat killed, or gamma irradiated Lactobacillus reuteri on cardio-autonomic response and single fibre unit discharge in dorsal root ganglia to colorectal distension in healthy Sprague-Dawley rats housed under conventional conditions. The effects of this treatment on somatic pain were also examined. Methods: 1×10 9 bacteria were given by gavage for nine days. Colorectal distension occurred under anaesthesia. Heart rate was measured through continuous electrocardiography. Single fibre unit discharge was recorded from the 6th left lumbar dorsal root ganglion. Somatic pain was evaluated by the tail flick and paw pressure tests. Results: Colorectal distension caused a pressure dependent bradycardia in the control (native medium) group. Treatment with live, heat killed, or gamma irradiated bacteria as well as their products (conditioned medium) prevented the pain response even during the maximum distension pressure (80 mm Hg). Both viable and non-viable bacteria significantly decreased dorsal root ganglion single unit activity to distension. No effects on somatic pain were seen with any treatment. Conclusions: Oral administration of either live or killed probiotic bacteria or conditioned medium inhibited the constitutive cardio-autonomic response to colorectal distension in rats through effects on enteric nerves. These data may provide a novel explanation for beneficial probiotic effects on visceral pain.

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.

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.

It is generally accepted that intestinal sensory vagal fibers are primary afferent, responding nonsynaptically to luminal stimuli. The gut also contains intrinsic primary afferent neurons (IPANs) that respond to luminal stimuli. A psychoactive Lactobacillus rhamnosus (JB-1) that affects brain function excites both vagal fibers and IPANs. We wondered whether, contrary to its primary afferent designation, the sensory vagus response to JB-1 might depend on IPAN to vagal fiber synaptic transmission. We recorded ex vivo single- and multiunit afferent action potentials from mesenteric nerves supplying mouse jejunal segments. Intramural synaptic blockade with Ca(2+) channel blockers reduced constitutive or JB-1-evoked vagal sensory discharge. Firing of 60% of spontaneously active units was reduced by synaptic blockade. Synaptic or nicotinic receptor blockade reduced firing in 60% of vagal sensory units that were stimulated by luminal JB-1. In control experiments, increasing or decreasing IPAN excitability, respectively increased or decreased nerve firing that was abolished by synaptic blockade or vagotomy. We conclude that >50% of vagal afferents function as interneurons for stimulation by JB-1, receiving input from an intramural functional "sensory synapse." This was supported by myenteric plexus nicotinic receptor immunohistochemistry. These data offer a novel therapeutic target to modify pathological gut-brain axis activity.-Perez-Burgos, A., Mao, Y.-K., Bienenstock, J., Kunze, W. A. The gut-brain axis rewired: adding a functional vagal nicotinic "sensory synapse."

The colocalization of three putative inhibitory mediators of enteric nerves, vasoactive intestinal peptide (VIP), galanin (GAL) and nitric oxide synthase (nNOS), was examined in the myenteric plexus of canine antrum, intestine and colon. Many ileal and colonic neurons contained nNOS-immunoreactive (nNOS-IR) activity with some also containing VIP-IR; only a few neurons also contained GAL-IR. Ileal and colonic VIP-IR nerves often appeared to be interneurons innervating nNOS nerves. Many antral neurons contained VIP-IR with nearly all also containing GAL-IR. A few also contained nNOS-IR. The predominance of nNOS-IR neurons relative to VIP-IR and GAL-IR neurons in the ileal and colonic, but not the antral, myenteric plexus is consistent with NO being the primary inhibitory mediator in the intestine but not in the antrum.

In canine ileum we investigated the distribution of pituitary adenylate cyclase-activating peptide (PACAP), using immunofluorescence and radioimmunoassay and the binding of125I-PACAP-27 to membranes. Nerve profiles immunoreactive to PACAP-27, and often to vasoactive intestinal polypeptide (VIP) as well, were found in all plexi, but PACAP was present in ∼100-fold lesser amounts than VIP. High-performance liquid chromatography analysis of deep muscular plexus (DMP) synaptosomes suggested the presence of PACAP-38, PACAP-27, and a third unidentified molecular form. High- and low-affinity125I-PACAP-27 binding sites were found in DMP synaptosomes and circular smooth muscle (CM) plasma membranes. In competition studies with DMP membranes, high (H)- and low (L)-affinity dissociation constants ( Kd) and maximal binding capacities (Bmax) were as follows: Kd H= 66.9 pM, Bmax H= 101 fmol/mg; Kd L= 2.18 nM, Bmax L= 580 fmol/mg protein. The binding of125I-PACAP-27 was fast. Dissociation was slow and incomplete in the presence of unlabeled PACAP-27 but accelerated by pretreatment with guanosine 5′- O-(3-thiotriphosphate) (GTPγS). GTPγS or cholera toxin treatment eliminated high-affinity binding in both membranes. VIP had ∼100-fold lower affinity than PACAP-27 in both membranes. Cross-linking studies identified an ∼70-kDa PACAP receptor in each membrane. Thus PACAP coexists with VIP in ileal enteric nerves and acts on PACAP-preferring, possibly Gs-coupled, receptors in DMP synaptosomes and CM membranes.

Enteric parasite infections around the world are a huge economic burden and decrease the quality of life for many people. The use of beneficial bacteria has attracted attention for their potential therapeutic applications in various diseases. However, the effects of beneficial bacteria in enteric parasitic infections remain largely unexplored. We investigated the effects of ingestion of Lactobacillus rhamnosus (JB-1) in a model of enteric nematode ( Trichuris muris ) infection. C57BL/6 (resistant to infection), AKR (susceptible to infection), interleukin 10 (IL-10) knockout (KO), and mucin Muc2 KO mice were infected with T. muris and treated orally with probiotic JB-1 or medium. The mice were sacrificed on various days postinfection to examine goblet cells, epithelial cell proliferation, cytokines, and worm burdens. Treatment with JB-1 significantly enhanced worm expulsion in resistant C57BL/6 mice, and this was associated with increases in IL-10 levels, goblet cell numbers, and epithelial cell proliferation. Beneficial effects of JB-1 were absent in IL-10 KO and resistant mice treated with γ-irradiated bacteria. Live JB-1 treatment also expedited worm expulsion in Muc2 KO mice and, more importantly, in AKR mice (susceptible to infection). Injection of IL-10 directly into the colonic tissue of uninfected mice induced goblet cell hyperplasia. These findings demonstrate that JB-1 modulates goblet cell biology and promotes parasite expulsion via an IL-10-mediated pathway and provide novel insights into probiotic effects on innate defense in nematode infection.

Symbionts or probiotics are known to affect the nervous system. To understand the mechanisms involved, it is important to measure sensory neuron responses and identify molecules responsible for this interaction. Here we test the effects of adding Lactobacillus rhamnosus (JB-1) and Bacteroides fragilis to the epithelium while making voltage recordings from intestinal primary afferent neurons. Sensory responses are recorded within 8 s of applying JB-1 and excitability facilitated within 15 min. Bacteroides fragilis produces similar results, as does its isolated, capsular exopolysaccharide, polysaccharide A. Lipopolysaccharide-free polysaccharide A completely mimics the neuronal effects of the parent organism. Experiments with a mutant Bacteroides fragilis devoid of polysaccharide A shows that polysaccharide A is necessary and sufficient for the neuronal effects. Complex carbohydrates have not been reported before as candidates for such signalling between symbionts and the host. These observations indicate new neuronal targets and invite further study of bacterial carbohydrates as inter-kingdom signalling molecules between beneficial bacteria and sensory neurons.

Background We have previously shown that ingestion of Lactobacillus reuteri may modulate colonic enteric neuron activity but with unknown effects on colon motility. The aim of the present report was to elucidate the neuronal mechanisms of action of the probiotic by comparing the effects on motility of L. reuteri ingestion with blockade of a specific ionic current in enteric neurons. Methods We have used intraluminal pressure recordings from ex vivo rat colon segments and whole cell patch clamp recordings from neurons of rat longitudinal muscle myenteric plexus preparations to investigate the effects of L. reuteri and TRAM-34 on colon motility and neurophysiology. The effects of daily feeding of 109L. reuteri bacteria or acute application of TRAM-34 on threshold fluid filling pressure or pulse pressure was measured. Key Results Lactobacillus reuteri increased intraluminal fluid filling pressure thresholds for evoking pressure pulses by 51% from 0.47 ± 0.17 hPa; the probiotic also decreased the pulse pressure amplitudes, but not frequency, by 18% from 3.91 ± 0.52 hPa. The intermediate conductance calcium-dependent potassium (IKCa) channel blocker TRAM-34 (3 μmol L−1) increased filling threshold pressure by 43% from 0.52 ± 0.22 hPa and reduced pulse pressure amplitude by 40% from 2.63 ± 1.11 hPa; contraction frequency was unaltered. TRAM-34 (3 μmol L−1) reduced membrane polarization, leak conductance and the slow afterhyperpolarization current in 16/16 myenteric rat colon AH cells but 19/19 S cells were unaffected. Conclusions & Inferences The present results are consistent with L. reuteri enhancing tonic inhibition of colon contractile activity by acting via the IKCa channel current in AH cells.

We report that neurons express functional Fc receptors (including FcεR1), that can be activated by antigen, transmit signals along nerve fibers in vitro and in vivo. These results open new avenues of investigation in neurogenic inflammation and allergic diseases.

Depolarization elicited outward K+currents from canine lower esophageal sphincter (LES) muscle cells, primarily through iberiotoxin (IbTX)- and tetraethylammonium-sensitive Ca2+-dependent K+channels. Current magnitudes varied with pipette Ca2+concentration (EC50= 108.5 nM). NG-nitro-l-arginine (l-NNA, 10−4M), IbTX (10−8M), or buffering intracellular Ca2+to 8 nM decreased outward currents >80%. Sodium nitroprusside (NaNP, 10−4M) restoredl-NNA-inhibited or low intracellular Ca2+concentration (not IbTX)-inhibited currents.l-NNA or IbTX application depolarized LES cells from −43 to −35 mV. NaNP restored the membrane potential to −46 mV afterl-NNA but not after IbTX application. Nifedipine (30 μM) reduced outward currents and abolished or reduced l-NNA or NaNP effects, respectively. Immunocytochemistry revealed the presence of both argininosuccinate synthetase and argininosuccinate lyase in LES muscle cells. l-Citrulline, likel-arginine, reversedl-NNA inhibition of outward currents; only l-arginine reversed inhibition of outward currents by an antibody to argininosuccinate synthetase. Therefore, endogenous nitric oxide production, activated by Ca2+entrance involving l-type Ca2+channels, may continuously enhance outward currents to modulate LES muscle cell membrane potential and excitability.

Wang, Y. F., Y. K. Mao, Q. Xiao, E. E. Daniel, K. R. Borkowski and T. J. McDonald. The distribution of Npy-containing nerves and the catecholamine contents of canine enteric nerve plexuses. Peptides 18(2) 221–234, 1997.—Our previous study demonstrated that PYY was a major neuropeptide in the canine enteric nervous system, the present study defines the locations of NPY-containing enteric neurons. NPY-positive nerve cell bodies and fibers were numerous in gastric and pyloric myenteric plexuses as were positive nerve fibers in antral and pyloric muscle layers, pyloric sphincter muscle layers, and surrounding blood vessels. In contrast to findings for PYY, there were considerably fewer NPY-positive nerve cell bodies and fibers in the canine ileum and colon. Noradrenaline was the predominant catecholamine in all plexuses, the rank order of its contents being: deep muscular > submucous > myenteric plexus. The dopamine/noradrenaline ratio was constant in all plexuses; adrenaline was present in minor amounts. PYY is the more abundant neuropeptide in ileum and colon, and NPY has a greater presence in the gastric antrum, pylorus, and surrounding blood vessels. PYY and NPY may play different functional roles in the GI tract.

PYY receptors were characterized and their loci determined in canine small intestine. The density of 125I-labeled peptide tyrosine tyrosine (PYY) binding was highest in myenteric (MY) and submucosal (SUB) plexus fractions enriched in synaptosomes. Two binding sites [high affinity (H) and low affinity (L)] were found in the submucosal synaptosome-enriched membrane: dissociation constant (Kd)H = 7.6 pM, maximal binding capacity (Bmax)H = 28 fmol/mg; KdL = 0.18 nM, BmaxL = 120 fmol/mg protein. The binding of 125I-PYY reached a maximum within 30 min; dissociation was incomplete in the presence of unlabeled PYY. The rate of dissociation was enhanced after exposure of synaptosomes to guanosine 5'-O-(3-thiotriphosphate). Binding of 125I-PYY was completely inhibited by neuropeptide Y (NPY)-(13-36) (in SUB and MY) and by [Leu31,Pro34]NPY (in MY) but only partially by [Leu31,Pro34]NPY in SUB, suggesting the presence of Y2 receptor in SUB and the presence of Y1 and Y2 receptors in MY. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of the PYY receptor complex revealed a radioactive band at 70 kDa. The PYY receptors in the canine small intestinal myenteric and submucosal plexus correspond in location to that of PYY in synaptosomes and are coupled with G proteins. Different subtypes are present in different loci.

Background: Vasoactive intestinal polypeptide (VIP) may be a nonadrenergic, noncholinergic inhibitory transmitter in the lower esophageal sphincter (LES). There is no biochemical evidence of VIP receptors in the LES. Methods: Using membranes from canine LES, VIP receptor distribution and characterization were analyzed by radioligand binding and cross-linking experiments. Results: High densities of saturable VIP receptors were found (maximum bound [Bmax], 539.2 fmol/mg in the synaptosome-enriched fraction [P2] and 732.7 fmol/mg in the smooth muscle, plasma membrane-enriched fraction [Mic II]), with high affinity for 125 I-VIP (dissociation constant [ K d ], 1.38 nmol/L in P2 and 1.40 nmol/L in Mic II). Competition binding studies suggested the presence of two binding sites, a high-affinity (inhibitor constant [ K 11 ], 0.064 nmol/L) and a low-affinity ( K 12 , 2.68 nmol/L) binding site in P2 membranes, but only one binding site ( K l , 1.18 nmol/ L) in Mic II membranes. Guanosine triphosphate-γ-s pretreatment eliminated high-affinity binding in P 2 membranes by conversion to binding sites of lower affinity ( K l , 2.82 nmol/L). Studies with a cross-linking agent identified VIP receptors in synaptosomal and smooth muscle plasma membrane fractions; a single polypeptide of approximately 60 kilodaltons was found in each membrane. Conclusions: Specific VIP receptors exist in both synaptosomal and smooth muscle plasma membrane of canine LES.