Yu-Kang Mao

Lactobacillus reuteri enhances excitability of colonic AH neurons by inhibiting calcium-dependent potassium channel opening

Probiotics are live non‐pathogenic commensal organisms that exert therapeutic effects in travellers’ diarrhea, irritable bowel syndrome and inflammatory bowel disease. Little is known about mechanisms of action of commensal bacteria on intestinal motility and motility‐induced pain. It has been proposed that probiotics affect intestinal nerve function, but direct evidence for this has thus far been lacking. We hypothesized that probiotic effects might be mediated by actions on colonic intrinsic sensory neurons. We first determined whether sensory neurons were present in rat colon by their responses to chemical mucosal stimulation and identified them in terms of physiological phenotype and soma morphotype. Enteric neuron excitability and ion channel activity were measured using patch clamp recordings. We fed 109Lactobacillus reuteri (LR) or vehicle control to rats for 9 days. LR ingestion increased excitability (threshold for evoking action potentials) and number of action potentials per depolarizing pulse, decreased calcium‐dependent potassium channel (IKCa) opening and decreased the slow afterhyperpolarization (sAHP) in sensory AH neurons, similar to the IKCa antagonists Tram‐34 and clotrimazole. LR did not affect threshold for action potential generation in S neurons. Our results demonstrate that LR targets an ion channel in enteric sensory nerves through which LR may affect gut motility and pain perception.

Inhibitory effects of Lactobacillus reuteri on visceral pain induced by colorectal distension in Sprague-Dawley rats

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: 1×109 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.

The microbiome is essential for normal gut intrinsic primary afferent neuron excitability in the mouse.

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.

Psychoactive bacteria Lactobacillus rhamnosus (JB-1) elicits rapid frequency facilitation in vagal afferents

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.

Lactobacillus reuteri ingestion prevents hyperexcitability of colonic DRG neurons induced by noxious stimuli

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.

Luminal administration ex vivo of a live Lactobacillus species moderates mouse jejunal motility within minutes

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 afterhy‐perpolarization (sAHP) by inhibiting a Ca‐activated K (IKCa) 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 IC50 of 5 × 107 cells/ml Krebs. Heat‐killed LR or another live commensal, Lactobacillus salivarius, were without effect. The IKCa 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.—Wang, B., Mao, Y.‐K., Diorio, C., Pasyk, M., Wu, R.Y., Bienenstock, J., Kunze, W. A. Luminal administration ex vivo of a live Lactobacillus species moderates mouse jejunal motility within minutes. FASEB J. 24, 4078–4088 (2010). www.fasebj.org

Bacteroides fragilis polysaccharide A is necessary and sufficient for acute activation of intestinal sensory neurons

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.

The gut microbiome restores intrinsic and extrinsic nerve function in germ-free mice accompanied by changes in calbindin.

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.

Evidence for neuronal expression of functional Fc (ε and γ) receptors

To the Editor: The Fc receptor family plays a key role in adaptive immunity through the binding of immunoglobulin antibodies that recognize an immune insult and elicit an inflammatory response leading to its clearance. Dysregulation of this receptor family may have untoward consequences that result in autoimmune and allergic diseases. Many of these diseases seem to involve the nervous system and are exacerbated by stress or other neurological challenges. Recently, the presence of Fc receptors was uncovered on dorsal root ganglion neurons and suggested an IgG and possibly IgE-mediated activation of neurons 1–3. We set out to more extensively explore which Fc receptors might be expressed in neurons, and whether they were functional and able to transmit signals to interconnected neurites in vitro and in vivo Messenger RNA was isolated from a highly pure culture of mouse superior cervical ganglion (SCG) neurons 4 and expression of Fc receptor transcripts assessed by reverse transcriptase-polymerase chain reactions (RT-PCR) using specific primers for Fcγ and Fce family members. Fig. 1,a demonstrates the presence of transcripts for the immunoglobulin-binding α chain of FcγRI, II, III, and IV in three individual SCG neuron mRNA preparations. A small amount of the transcript for the low affinity IgE receptor (FceRII or CD23) was also detected relative to that seen in B cells, known to express this receptor. FcγRI transcripts were detected in bone marrow derived mouse mast cells but these levels were less than seen in the neurons. This observation and the inability to detect mMCP-6mRNA in either Balb/c or Bl6 mice, together with the absence of CD23 transcripts in both neurons and mast cells provided confidence that the observed Fc receptor transcripts in neurons was not a result of mast cell contamination of cultures. Figure 1 mRNA expression of the subunits of FceRI, FcγRI-IV, and CD23 in SCG neurons. mRNA was collected from three (1–3) SCG neuronal cultures and expression of the indicated mRNA was measured by RT-PCR. (a) Expression of the α ... We also unexpectedly observed the presence of transcripts for the α, β, and γ chains of the high affinity IgE receptor (FceRI) (Fig. 1,b). While the trimeric form (αγ2) of this receptor has been described in cells other than mast cells or basophils (such as in human Langerhans cells 5), the expression of the tetrameric form (αβγ2) was previously thought to be limited to these pro-inflammatory cells. The trimeric FceRI shows weak calcium signals when compared to the tetrameric form due to the absence of the FceRIβ in the former 6. To determine if the FceRI was expressed on the cell surface of SCG neurons, cells were incubated with IgE and with an antibody to the neuronal specific protein gene product (PGP) 9.5 (which encodes a neuronal ubiquitin C-terminal hydrolase not found on glia) and binding visualized with a fluorescent secondary antibody. IgE was detected on the neuronal cell surface (Fig. 2,a). While the presence of PGP9.5 was most evident in the cell body, IgE binding was detected in plasma membrane of both the cell body and neurite extensions. As shown in Figs. 2,b–d, the expression of FceRI was further confirmed by detection of the α, β, and γ chains of this receptor. Figure 2 Neurons were sensitized with IgE and incubated overnight with antibody against IgE (a, left panel) or with an antibody of unknown specificity (a, right panel). Staining with the neuron specific marker PGP9.5 (b–c, left panel in red) and FceRIα ... Scorpion venom is a known potent selective activator of neurons and elicits a rapid rise in intracellular Ca2+ (Fig. 3,a). To test the functionality of FceRI expressed on neurons, SCG neurons sensitized with DNP-specific IgE were challenged with DNP-HSA (Ag) and a rapid rise in intracellular Ca2+ was observed (Fig. 3,a). No changes in intracellular Ca2+ were observed when serum albumin alone was used as Ag or when cells were not sensitized with IgE (data not shown). FcγRIV was recently described to bind the IgEb allotype but does not recognize the IgEa allotype 7, whereas FceRI binds both allotypes. We excluded that IgE/Ag-mediated calcium responses might occur through FcγRIV, by use of both a IgEa and IgEb allotypes. Both IgE allotypes similarly elicited calcium responses (Fig. 3,a). Addition of Ag elicited a relatively uniform Ca2+ responses in the stimulated cell population (Fig. 3,b). . Based on the high affinity binding of monomeric IgE (was not removed by washing the cells) and the dose response to Ag (Fig. 3, D), we could also exclude the involvement of CD23. Figure 3 (a) Kinetics of [Ca2+]i increase upon stimulation with scorpion venom (SV, Control) or with anti-DNP IgEa,/b sensitized neurons following Ag stimulation (10 ng/ml). The arrows indicate the time of addition of the stimulus. (b) Confocal image of calcium ... Thus, the findings demonstrate the presence of functional high affinity Fce receptors on SCG neurons. Since FcγRIII is known to activate mast cells, its functionality was also tested. SCG neurons sensitized with DNP-specific IgG1, which preferentially binds FcγRII and III but weakly to FcγRI and not to FcγRIV 8, showed modest increases in intracellular Ca2+ that increased with a large dose of Ag (Fig. 3,c). Both IgE and IgG-mediated responses were concentration dependent and a Ca2+ response (Fig. 3,d) was not elicited in all challenged neurons. Moreover, as expected (given the weak binding of monomeric IgG1 to Fcγ receptors), increased responsiveness via IgG required much higher concentrations of Ag than for IgE. To test if the Ca2+ signals elicited by FceRI stimulation could be transferred to interconnected neurites, we explored whether Ca2+ rises might be elicited in neighboring neurites after Ag challenge of an IgE sensitized cell body or neurite. Using a spritzer micro-pipette, antigen was puffed directly onto a neuronal cell body, causing an instantaneous (<5 sec) increased fluorescence in that cell body which moved from there to the connected neurites and propagated to the neighboring cell bodies and neurites (Fig. 4). These, findings showed that FceRI stimulation causes communication among interconnected neurites. To extend these findings to a more physiological setting, we explored whether neurons from the highly innervated intact jejunum 9 would respond to an FceRI stimulus. After placement of a micropipette spritzer (dotted lines shown in Fig. 5,a and g) on a large myenteric plexus ganglion neuron (plain lines shown in Fig. 5,a and g), the anti-DNP IgE sensitized plexus was challenged with Ag. Challenge with a spritz of 1 μg of Ag gave robust calcium responses in adjacent neurons along the nerve fiber in sensitized (2/2) but not in non-sensitized mice (0/3). Repeated spritzes of non-conjugated HSA at this concentration elicited no responses (0/2) (data not shown). To exclude possible mast cell involvement in the transmission of these robust signals, we conducted similar experiments in mast cell deficient W/Wv mice and in their wild type control WBB6F1. In W/Wv mice (3/3) detectable intracellular calcium increases were observed upon Ag challenge (Fig. 5,a–c). No calcium signal was seen when the same ganglion was first challenged with HSA alone (Fig. 5,d–f). The wild type littermates (WBB6F1) responded positively (3/3), upon challenge with specific Ag (Fig. 5,g–i) but gave no response to HSA alone. These findings confirm that the observed signal transmission by FceRI was not likely caused by mast cells and demonstrate the in vivo presence of functional FceRI on jejunal neurons, since sham sensitization in vivo prior to an ex vivo challenge yielded no response to Ag challenge. Figure 4 Activation of interconnected neurites by FceRI stimulation of a single neuronal cell body. (a) Bright field image of neuron network showing relation of Ag-containing spritzer to neuronal cell body sensitized with IgE anti-DNP. (b) At time zero, ... Figure 5 (A–I) Myenteric ganglion calcium imaging. Spritzer (internal bore, 40 μm) is indicated by dotted lines and myenteric plexus by solid lines. (a–c) Anti-DNP IgE sensitized myenteric neurons were imaged in mast cell-deficient W/W ... It is well known that sensory nerves may participate in hypersensitivity reactions, a process known as neurogenic inflammation and several lines of evidence support the notion that sensory nerves may play an important role in cutaneous, lung, GI and joint inflammatory diseases. Here we now demonstrate that functionally active FceRI is expressed on SCG and myenteric plexus neurons. The discovery of functional Fce and Fcγ receptors on nerves clearly shows that this biological compartment is able to respond to the direct stimulus of antibody-antigen interactions. Our findings define an independent neuronal (non-mast cell/non-basophil) compartment with probable involvement in allergic and possibly other diseases.

Spatiotemporal maps reveal regional differences in the effects on gut motility for Lactobacillus reuteri and rhamnosus strains

Background  Commensal bacteria such as probiotics that are neuroactive acutely affect the amplitudes of intestinal migrating motor complexes (MMCs). What is lacking for an improved understanding of these motility effects are region specific measurements of velocity and frequency. We have combined intraluminal pressure recordings with spatiotemporal diameter maps to analyze more completely effects of different strains of beneficial bacteria on motility.