Differences in cecal microbiome of selected high and low feather-pecking laying hens.

In mammals, it has become increasingly clear that the gut microbiota influences not only gastrointestinal physiology but also modulates behavior. In domestic birds, ceca have the greatest gastrointestinal microbial population. Feather-pecking (FP) behavior in laying hens is one of the most important unsolved behavioral issues in modern agriculture. The aim of the present study was to assess the cecal microbial community of divergently selected high (HFP; n = 20) and low (LFP; n = 20) feather-pecking birds at 60 wk of age. The cecal samples were subjected to community profiling of 16S rRNA and in silico metagenomics using a modified bar-coded Illumina sequencing method on a MiSeq Illumina sequencer. Our results revealed that compared to HFP birds, LFP birds are characterized by an increased overall microbial diversity (beta diversity) shown by a difference in the Bray-Curtis index (R2 = 0.171, P < 0.05). Furthermore, operational taxonomic unit comparisons showed an increased presence of Clostridiae and decreased presence of Lactobaccillacae in HFP birds when compared to LFP birds (False Discovery Rate < 0.05, Mann-Whitney comparisons). Our data indicate that there may be differences in the cecal profile between these 2 lines of laying hens. More research, building on this first study using sequencing technology for profiling the chicken cecal microbiome, will be needed in order to reveal if and how there exists a functional link between the performance of FP and the cecal microbial community.

Lactobacillus rhamnosus strain JB-1 reverses restraint stress-induced gut dysmotility.

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.

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

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.

Oxytocin-induced membrane hyperpolarization in pain-sensitive dorsal root ganglia neurons mediated by Ca(2+)/nNOS/NO/KATP pathway.

Oxytocin (OT) plays an important role in pain modulation and antinociception in the central nervous system. However, little is known about its peripheral effects. This study was conducted to investigate the effect of OT on the electrical properties of neurons in the dorsal root ganglia (DRG) and the underlying mechanisms. DRG neurons from adult rats were acutely dissociated and cultured. Intracellular Ca(2+) was determined by fluorescent microscopy using an indicator dye. The electrical properties of DRG neurons were tested by patch-clamp recording. The oxytocin receptor (OTR) and neuronal nitric oxide synthase (nNOS) on DRG neurons were assessed with immunofluorescence assays. OTR co-localized with nNOS in most of Isolectin B4 (IB4)-binding cultured DRG neurons in rats. OT decreased the excitability, increased the outward current, and evoked the membrane hyperpolarization in cultured DRG neurons. Sodium nitroprusside (SNP), the donor of nitric oxide (NO), exerted similar effects as OT on the membrane potential of cultured DRG neurons. OT increased the production of NO in DRGs and cultured DRG neurons. Pre-treatment of the OTR antagonist atosiban or the selective nNOS inhibitor N-Propyl-l-arginine (NPLA) significantly attenuated the hyperpolarization effect evoked by OT. OT produced a concentration-dependent increase in intracellular Ca(2+) in DRG neurons that responds to capsaicin, which can be attenuated by atosiban, but not by NPLA. OT-evoked membrane hyperpolarization and increase of outward current were distinctly attenuated by glibenclamide, a blocker of ATP-sensitive K(+) (KATP) channel. OT might be an endogenous antinociceptive agent and the peripheral antinociceptive effects of OT are mediated by activation of the Ca(2+)/nNOS/NO/KATP pathway in DRG neurons.

Oxytocin down-regulates mesenteric afferent sensitivity via the enteric OTR/nNOS/NO/KATP pathway in rat.

BACKGROUND: Oxytocin plays an analgesic role in modulation of nociception and pain. Most work to date has focused on the central mechanisms of oxytocin analgesia, but little is known about whether peripheral mechanisms are also involved. METHODS: The mesenteric afferent discharge was recorded in vitro. The expressions of oxytocin receptor (OTR) and neuronal nitric oxide synthase (nNOS) in longitudinal muscle myenteric plexus (LMMP) was identified by immunofluorescence. KEY RESULTS: Oxytocin per se had no effect on the jejunal mesenteric afferent discharge, however, it markedly attenuated the bradykinin- or distention-evoked increase of mesenteric afferent discharge, which was mimiced by the nitric oxide (NO) donor sodium nitroprusside (SNP). Pretreatment of either NOS inhibitor L-NAME or NPLA largely reduced the inhibitory effect of oxytocin on bradykinin-evoked mesenteric afferent discharge. Such effect, to a large extent, was also alleviated by N-and P-type voltage-dependent calcium channel antagonists or KATP blocker glibenclamide. In addition, immunofluorescence studies show strong colocalization of OTR with nNOS in LMMP of the rat jejunum. CONCLUSIONS & INFERENCES: Oxytocin down-regulates the mesenteric afferent sensitivity through nNOS-NO-KATP pathway. Our findings may reveal a new peripheral mechanism for oxytocin analgesia.

Bifidobacterium longum NCC3001 inhibits AH neuron excitability.

BACKGROUND: Bifidobacterium longum (B. longum) NCC3001 can affect behavior and brain biochemistry, but identification of the cellular targets needs further investigation. Our hypothesis was that the communication with the brain might start with action on enteric sensory neurons. METHODS: Ileal segments from adult mice were used to create a longitudinal muscle-myenteric-plexus preparation to expose sensory after-hyperpolarizing (AH) neurons in the myenteric plexus to allow access with microelectrodes. The intrinsic excitability of AH neurons was tested in response to the perfusion of conditioned media (B. longum culture supernatant) or unconditioned media (growth medium, MRS). KEY RESULTS: B. longum conditioned medium significantly reduced the excitability of AH neurons compared to perfusion with the unconditioned medium. Specifically, a reduction was seen in the number of action potentials fired per depolarizing test pulse, the instantaneous and time-dependent input resistances and the magnitude of the hyperpolarization-activated cationic current (Ih ). CONCLUSIONS & INFERENCES: The probiotic B. longum reduces excitability of AH sensory neurons likely via opening of potassium channels and closing of hyperpolarization-activated cation channels.

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. METHODS: Intraluminal peak pressure (PPr) was measured and video recordings made of mouse ex vivo jejunum and colon segments before and after intraluminal applications of Lactobacillus rhamnosus (JB-1) or Lactobacillus reuteri (DSM 17938). Migrating motor complex frequency and velocity were calculated. KEY RESULTS: JB-1 decreased jejunal frequencies by 56% and 34% in colon. Jejunal velocities increased 171%, but decreased 31% in colon. Jejunal PPr decreased by 55% and in colon by 21%. DSM 17938 increased jejunal frequencies 63% and in colon 75%; jejunal velocity decreased 57%, but increased in colon 146%; jejunal PPr was reduced 26% and 12% in colon. TRAM-34 decreased frequency by 71% and increased velocity 200% for jejunum, but increased frequency 46% and velocity 50% for colon; PPr was decreased 59% for jejunum and 39% for colon. CONCLUSIONS & INFERENCES: The results show that probiotics and other beneficial bacteria have strain and region-specific actions on gut motility that can be successfully discriminated using spatiotemporal mapping of diameter changes. Effects are not necessarily the same in colon and jejunum. Further research is needed on the detailed effects of the strains on enteric neuron currents for each gut region.

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. 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.

The microbiota and the gut-brain axis: insights from the temporal and spatial mucosal alterations during colonisation of the germfree mouse intestine.

The influence of the gut microbiota on the nervous system, brain development and behaviour, in particular during microbial colonisation of the host, has recently been receiving profound interest. Our time-resolved mining of combined data analyses of the ex-germfree mouse intestine during a 30-day course of colonisation with conventional mouse faecal microbiota (conventionalisation), shed light on temporal altered expression of genes of which the products influenced functions of the nervous system. Plasma tryptophan and kynurenine levels reflected high indoleamine dioxygenase activity, which was supported by significant temporal induction of the encoding gene in all gut tissues. However, the majority of genes associated with neuronal development and function were reduced. Colonic substance P elevation in response to conventionalisation was higher only after 30-days. These results support a functional microbiota-neurohumoral relationship during conventionalisation and suggest a delayed neuronal response that is elicited only after the microbiota accommodating homeostasis has been accomplished.

The anxiolytic effect of Bifidobacterium longum NCC3001 involves vagal pathways for gut-brain communication.

BACKGROUND: The probiotic Bifidobacterium longum NCC3001 normalizes anxiety-like behavior and hippocampal brain derived neurotrophic factor (BDNF) in mice with infectious colitis. Using a model of chemical colitis we test whether the anxiolytic effect of B. longum involves vagal integrity, and changes in neural cell function. Methods Mice received dextran sodium sulfate (DSS, 3%) in drinking water during three 1-week cycles. Bifidobacterium longum or placebo were gavaged daily during the last cycle. Some mice underwent subdiaphragmatic vagotomy. Behavior was assessed by step-down test, inflammation by myeloperoxidase (MPO) activity and histology. BDNF mRNA was measured in neuroblastoma SH-SY5Y cells after incubation with sera from B. longum- or placebo-treated mice. The effect of B. longum on myenteric neuron excitability was measured using intracellular microelectrodes. KEY RESULTS: Chronic colitis was associated with anxiety-like behavior, which was absent in previously vagotomized mice. B. longum normalized behavior but had no effect on MPO activity or histological scores. Its anxiolytic effect was absent in mice with established anxiety that were vagotomized before the third DSS cycle. B. longum metabolites did not affect BDNF mRNA expression in SH-SY5Y cells but decreased excitability of enteric neurons. CONCLUSIONS & INFERENCES: In this colitis model, anxiety-like behavior is vagally mediated. The anxiolytic effect of B. longum requires vagal integrity but does not involve gut immuno-modulation or production of BDNF by neuronal cells. As B. longum decreases excitability of enteric neurons, it may signal to the central nervous system by activating vagal pathways at the level of the enteric nervous system.

Lactobacillus reuteri ingestion and IK(Ca) channel blockade have similar effects on rat colon motility and myenteric neurones.

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 10(9) L. 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 (IK(Ca)) channel blocker TRAM-34 (3 micromol 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 micromol 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 IK(Ca) channel current in AH cells.

Recurrent posttraumatic meningitis due to nontypable Haemophilus influenzae: case report and review of the literature.

We report a case of relapsing Haemophilus influenzae meningitis in a boy at the age of nearly 3 years and 4.2 years who had been successfully vaccinated against H. influenzae serotype b (Hib). The pathogen was a nonencapsulated (nontypable) H. influenzae strain of biotypes III and VI, respectively. A rhinobasal impalement injury with development of a posttraumatic encephalocele is considered to be the predisposing condition. Review of the literature reveals that in patients systemically infected by nonencapsulated H. influenzae strains predisposing factors such as cerebrospinal fluid-shunts, implants and traumas are often found. To obtain further information on potential new disease patterns H. influenzae isolates from cerebrospinal fluid should be examined for capsule production and, if relevant, further characterized by capsular typing.

Nitric oxide mediates the inhibitory effect of ethanol on the motility of isolated longitudinal muscle of proximal colon in rats.

The aim of the present study was to investigate the effect of ethanol on colon motility in rats and to test the possibility that nitric oxide (NO) mediates this effect. Proximal colon longitudinal muscle strips (LM) (8 x 3 mm) cut parallel to the longitudinal muscle fibres of the colon were isolated and mounted in an organ bath. Ethanol (0.57, 0.87 and 1.30 mmol L(-1)) dose-dependently inhibited the motility of LM. Longitudinal muscle strips from female rats were more sensitive to the inhibitory effect of ethanol than that from male rats. L-NAME (N-nitro-L-arginine methyl ester) (100 micromol L(-1)), AG (aminoguanidine) (10 micromol L(-1)), ODQ (1H-[1,2,4]Oxadiazolo[4,3-a]quinoxalin-1-one) (10 micromol L(-1)) and PTIO (2-Phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide) (200 micromol L(-1)) partly blocked the inhibitory effect of ethanol on LM. Pretreatment with L-NAME, AG, ODQ and PTIO abolished the sex difference of the inhibitory effect of ethanol on LM. Tetrodotoxin (TTX) (10 micromol L(-1)) partly blocked the inhibitory effect but did not influence the sex difference. The relaxation of LM induced by SNP (sodium nitroprusside) (0.1-10 micromol L(-1)) in female rats was greater than that in male rats. In conclusion, ethanol inhibited the colon motility in vitro. This inhibitory effect on LM was mediated by NO through the iNOS - NO - cGMP pathway.

Responses of myenteric S neurones to low frequency stimulation of their synaptic inputs.

Previous experiments have shown that prolonged low frequency stimulation of presynaptic inputs causes excitation of AH neurones that considerably outlasts the period of stimulation in the guinea-pig small intestine. The present experiments compare the responses of S neurones (which are motor neurones and interneurones) with responses of AH neurones (intrinsic primary afferent neurones) to low frequency stimulation of synaptic inputs. Neurones in the myenteric plexus of isolated segments of guinea-pig small intestine were recorded from with intracellular microelectrodes. During their impalement, the neurones were filled with a marker dye and they were later processed to reveal their shapes and immunohistochemical properties. One group of neurones, inhibitory motor neurones to the circular muscle, was depolarised by stimulation of synaptic inputs at 1 Hz for 100 s to 4 min. With 4-min trains of stimuli, peak depolarisation was 21+/-2 mV (mean+/-S.E.M.), which was reached at about 110 s. Depolarisation was accompanied by increased excitability; before stimulation, a test intracellular pulse (500 ms) triggered 3 action potentials, at the peak of excitability this reached 16 action potentials. Depolarisation began to decline immediately at the end of stimulation. This contrasts with responses of AH neurones, in which depolarisation persisted after the end of the stimulus (peak depolarisation at 300 s). The excitation and depolarisation of inhibitory motor neurones was blocked by the neurokinin 1 tachykinin receptor antagonist, SR140333 (100 nM), but excitation of AH neurones was not affected. Small or no responses to 1 Hz stimulation were recorded from descending filamentous interneurones, longitudinal muscle motor neurones and excitatory circular muscle motor neurones. In conclusion, this study indicates that sustained slow postsynaptic excitation only occurs in AH neurones, and that one type of S neurones, inhibitory motor neurones to the circular muscle, responds substantially, but not beyond the period of stimulation, to activation of synaptic inputs at 1 Hz. This slow excitatory postsynaptic potential evoked by low frequency stimulation is mediated by tachykinins.

Afterhyperpolarization current in myenteric neurons of the guinea pig duodenum.

Whole cell patch and cell-attached recordings were obtained from neurons in intact ganglia of the myenteric plexus of the guinea pig duodenum. Two classes of neuron were identified electrophysiologically: phasically firing AH neurons that had a pronounced slow afterhyperpolarization (AHP) and tonically firing S neurons that lacked a slow AHP. We investigated the properties of the slow AHP and the underlying current (I(AHP)) to address the roles of Ca(2+) entry and Ca(2+) release in the AHP and the characteristics of the K(+) channels that are activated. AH neurons had a resting potential of -54 mV and the AHP, which followed a volley of three suprathreshold depolarizing current pulses delivered at 50 Hz through the pipette, averaged 11 mV at its peak, which occurred 0.5-1 s following the stimulus. The duration of these AHPs averaged 7 s. Under voltage-clamp conditions, I(AHP)'s were recorded at holding potentials of -50 to -65 mV, following brief depolarization of AH neurons (20-100 ms) to positive potentials (+35 to +50 mV). The null potential of the I(AHP) at its peak was -89 mV. The AHP and I(AHP) were largely blocked by omega-conotoxin GVIA (0.6-1 microM). Both events were markedly decreased by caffeine (2-5 mM) and by ryanodine (10-20 microM) added to the bathing solution. Pharmacological suppression of the I(AHP) with TEA (20 mM) or charybdotoxin (50-100 nM) unmasked an early transient inward current at -55 mV following step depolarization that reversed at -34 mV and was inhibited by niflumic acid (50-100 microM). Mean-variance analysis performed on the decay of the I(AHP) revealed that the AHP K(+) channels have a mean chord conductance of ~10 pS, and there are ~4,000 per AH neuron. Spectral analysis showed that the AHP channels have a mean open dwell time of 2.8 ms. Cell-attached patch recordings from AH neurons confirmed that the channels that open following action currents have a small unitary conductance (10-17 pS) and open with a high probability (

Comparison of the effects of neurokinin-3 receptor blockade on two forms of slow synaptic transmission in myenteric AH neurons.

AH neurons are intrinsic sensory neurons of the intestine that exhibit two types of slow synaptic event: slow excitatory postsynaptic potentials which increase their excitability for about 2-4 min, and sustained slow postsynaptic excitation which can persist for several hours, and may be involved in long-term changes in the sensitivity of the intestine to sensory stimuli. The effects of the neurokinin-3 tachykinin receptor antagonist, SR142801, on these two types of synaptic event in AH neurons of the myenteric ganglia of guinea-pig small intestine were compared. Slow excitatory postsynaptic potentials were evoked by stimulation of synaptic inputs at 10-20 Hz for 1s, and sustained slow postsynaptic excitation was evoked by stimulation of inputs at 1Hz for 4 min. SR142801 (1microM) reduced the amplitude of the slow excitatory postsynaptic potential to 26% of control, and also reduced the increase in input resistance and the extent of anode break excitation associated with the slow excitatory postsynaptic potential. In contrast, SR142801 did not reduce the increase in excitability, the increase in input resistance or the depolarisation that occur during the sustained slow postsynaptic excitation. SR142801 did not change the resting membrane potential or the resting input resistance. We conclude that tachykinins, acting through neurokinin-3 receptors, are involved in the generation of the slow excitatory postsynaptic potential, but not in the sustained slow postsynaptic excitation, and that the release of transmitters from synaptic inputs to AH neurons is frequency coded.

The terminals of myenteric intrinsic primary afferent neurons of the guinea-pig ileum are excited by 5-hydroxytryptamine acting at 5-hydroxytryptamine-3 receptors.

The aim of this study was to identify the receptor type(s) by which 5-hydroxytryptamine applied to the intestinal mucosa excites the terminals of myenteric AH neurons. The AH neurons have been identified as the intrinsic primary afferent (sensory) neurons in guinea-pig small intestine and 5-hydroxytryptamine has been identified as a possible intermediate in the sensory transduction process. Intracellular recordings were taken from AH neurons located within 1mm of intact mucosa to which 5-hydroxytryptamine was applied. Trains of action potentials and/or slow depolarizing responses were recorded in AH neurons in response to mucosal application of 5-hydroxytryptamine (10 or 20microM) or the 5-hydroxytryptamine-3 receptor agonist, 2-methyl-5-hydroxytryptamine (1 or 3mM), and to electrical stimulation of the mucosa. The 5-hydroxytryptamine-2 receptor agonist, alpha-methyl-5-hydroxytryptamine (100microM), and the 5-hydroxytryptamine-1,2,4 receptor agonist, 5-methoxytryptamine (10microM), did not elicit such responses. The 5-hydroxytryptamine-3 receptor-selective antagonist, granisetron (typically 1microM), and the 5-hydroxytryptamine-3,4 receptor antagonist, tropisetron (typically 1microM), each reduced or abolished the responses to 5-hydroxytryptamine, while the selective 5-hydroxytryptamine-4 receptor antagonist, SB 204070 (1microM), did not. It is concluded that application of 5-hydroxytryptamine to the mucosa activates a 5-hydroxytryptamine-3 receptor that triggers action potential generation in the mucosal nerve terminals of myenteric AH neurons.

Shapes and projections of tertiary plexus neurons of the guinea-pig small intestine.

The axons of neurons that innervate the longitudinal muscle of the small intestine in small mammals such as rabbit, rat, guinea pig and mouse form a network, the tertiary plexus, against the inner surface of the muscle. In general, because of their substantial overlap, it has not been possible to follow the ramifications of individual axons in the tertiary plexus. In the present work, the longitudinal muscle motor neurons were filled with marker dyes through an intracellular microelectrode, and their morphologies and projections were examined in whole-mount preparations of longitudinal muscle and myenteric plexus. Most neurons that were examined were in the small intestine (ileum and duodenum), but a few were examined in the distal colon. Neurons in all regions had similar morphologies and projections. The cell bodies were amongst the smallest in myenteric ganglia, with major and minor axes of 14 microns and 25 microns (mean, n = 40) in the plane of the myenteric plexus. Each neuron had a single axon that branched extensively in the tertiary plexus, most had multiple lamellar dendrites and a few had filamentous dendrites or a mixture of filamentous and lamellar dendrites. The mean area of muscle covered by an axon and its branches extended 1.6 mm orally to anally and 1.7 mm circumferentially. The area covered was 2.8 +/- 1.9 mm2 (mean +/- SD, n = 23). From the density of occurrence of cell bodies, it can be calculated that each point in the longitudinal muscle is innervated by the processes of about 100 motor neurons and is influenced by electrotonic conduction of signals through the muscle by about 300 motor neurons.

The soma and neurites of primary afferent neurons in the guinea-pig intestine respond differentially to deformation.

1. Intrinsic primary afferent neurons in the small intestine are exposed to distortion of their processes and of their cell bodies. Recordings of mechanosensitivity have previously been made from these neurons using intracellular microelectrodes, but this form of recording has not permitted detection of generator potentials from the processes, or of responses to cell body distortion. 2. We have developed a technique to record from enteric neurons in situ using patch electrodes. The mechanical stability of the patch recordings has allowed recording in cell-attached and whole cell configuration during imposed movement of the neurons. 3. Pressing with a fine probe initiated generator potentials (14 +/- 9 mV) from circumscribed regions of the neuron processes within the same myenteric ganglion, at distances from 100 to 500 microm from the cell body that was patched. Generator potentials persisted when synaptic transmission was blocked with high Mg2+, low Ca2+ solution. 4. Soma distortion, by pressing down with the whole cell recording electrode, inhibited action potential firing. Consistent with this, moderate intra-electrode pressure (10 mbar; 1 kPa) increased the opening probability of large-conductance (BK) potassium channels, recorded in cell-attached mode, but suction was not effective. In outside-out patches, suction, but not pressure, increased channel opening probability. Mechanosensitive BK channels have not been identified on other neurons. 5. The BK channels had conductances of 195 +/- 25 pS. Open probability was increased by depolarization, with a half-maximum activation at a patch potential of 20 mV and a slope factor of 10 mV. Channel activity was blocked by charybdotoxin (20 nM). 6. Stretch that increased membrane area under the electrode by 15 % was sufficient to double open probability. Similar changes in membrane area occur when the intestine changes diameter and wall tension under physiological conditions. Thus, the intestinal intrinsic primary afferent neurons are detectors of neurite distortion and of compression of the soma, these stimuli having opposite effects on neuron excitability.