α-Synuclein Regulates Development and Function of Cholinergic Enteric Neurons in the Mouse Colon.

Alpha-Synuclein (α-Syn) is expressed in the central nervous system and the nervous system of the gut (enteric nervous system, ENS), and is well known to be the major constituent of Lewy bodies which are the hallmark of Parkinson's disease. Gastrointestinal disorders frequently manifest several years before motor deficits develop in Parkinson's patients. Despite extensive research on pathological rodent models, the physiological role of α-Syn in the normal ENS is unclear hampering analysis of its neuropathology. We compared the ENS in colons of α-Syn-knockout (α-Syn KO) and wild-type mice using immunohistochemistry and calcium-imaging of responses to synaptic input. We found that α-Syn is predominantly expressed in cholinergic varicosities, which contain vesicular acetylcholine transporter. α-Syn KO mice had higher enteric neuron density and a larger proportion of cholinergic neurons, notably those containing calretinin, demonstrating a role for α-Syn in regulating development of these neurons. Moreover, α-Syn deletion enhanced the amplitude of synaptically activated [Ca2+]i transients that are primarily mediated by acetylcholine activating nicotinic receptors suggesting that α-Syn modulates the availability of acetylcholine in enteric nerve terminals.

Targeting eotaxin-1 and CCR3 receptor alleviates enteric neuropathy and colonic dysfunction in TNBS-induced colitis in guinea pigs.

BACKGROUND: The accumulation of eosinophils is mediated by the chemokine receptor-3 (CCR3)-eotaxin axis. Increased expression of eotaxin and its receptor is associated with inflammatory bowel disease (IBD). Activation of eosinophils causes the release of cationic proteins that are neurotoxic such as eosinophil-derived neurotoxin (EDN). Damage to enteric neurons alters neurally controlled functions of the gut correlated with intestinal inflammation. We hypothesized that inhibition of the CCR3-eotaxin axis will prevent inflammation-induced functional changes to the gastrointestinal tract. METHODS: Hartley guinea pigs were administered with trinitrobenzene sulfonate (TNBS; 30 mg/kg in 30% ethanol) intrarectally to induce colitis. A CCR3 receptor antagonist (SB 328437 [SB3]) was injected intraperitoneally 1 hour postinduction of colitis. Animals were euthanized 7 days post-treatment and colon tissues were collected for ex vivo studies. The EDN-positive eosinophils in the colon, indicating eosinophil activation, were quantified by immunohistochemistry. Effects of SB3 treatment on gross morphological damage, enteric neuropathy, and colonic dysmotility were determined by histology, immunohistochemistry, and organ bath experiments. KEY RESULTS: The number of EDN-positive eosinophils was significantly increased in the lamina propria in close proximity to myenteric ganglia in inflamed colon. The TNBS-induced inflammation caused significant damage to colonic architecture and inhibition of colonic motility. Treatment with SB3 antagonist attenuated inflammation-associated morphological damage in the colon, reduced infiltration of EDN-positive eosinophils and restored colonic motility to levels comparable to control and sham-treated guinea pigs. CONCLUSION & INFERENCES: This is the first study demonstrating that inhibition of CCR3-eotaxin axis alleviates enteric neuropathy and restores functional changes in the gut associated with TNBS-induced colitis.

Gastrointestinal dysfunction and enteric neurotoxicity following treatment with anticancer chemotherapeutic agent 5-fluorouracil.

BACKGROUND: The use of the anticancer chemotherapeutic agent 5-fluorouracil (5-FU) is often limited by nausea, vomiting, constipation, and diarrhea; these side-effects persist long after treatment. The effects of 5-FU on enteric neurons have not been studied and may provide insight into the mechanisms underlying 5-FU-induced gastrointestinal dysfunction. METHODS: Balb/c mice received intraperitoneal injections of 5-FU (23 mg/kg) 3 times/week for 14 days. Gastrointestinal transit was analysed in vivo prior to and following 3, 7, and 14 days of 5-FU treatment via serial x-ray imaging. Following 14 days of 5-FU administration, colons were collected for assessment of ex vivo colonic motility, gross morphological structure, and immunohistochemical analysis of myenteric neurons. Fecal lipocalin-2 and CD45+ leukocytes in the colon were analysed as markers of intestinal inflammation. KEY RESULTS: Short-term administration of 5-FU (3 days) increased gastrointestinal transit, induced acute intestinal inflammation and reduced the proportion of neuronal nitric oxide synthase-immunoreactive neurons. Long-term treatment (7, 14 days) resulted in delayed gastrointestinal transit, inhibition of colonic migrating motor complexes, increased short and fragmented contractions, myenteric neuronal loss and a reduction in the number of ChAT-immunoreactive neurons after the inflammation was resolved. Gross morphological damage to the colon was observed following both short- and long-term 5-FU treatment. CONCLUSIONS & INFERENCES: Our results indicate that 5-FU induces accelerated gastrointestinal transit associated with acute intestinal inflammation at day 3 after the start of treatment, which may have led to persistent changes in the ENS observed after days 7 and 14 of treatment contributing to delayed gastrointestinal transit and colonic dysmotility.

Enteric nervous system assembly: Functional integration within the developing gut.

Co-ordinated gastrointestinal function is the result of integrated communication between the enteric nervous system (ENS) and "effector" cells in the gastrointestinal tract. Unlike smooth muscle cells, interstitial cells, and the vast majority of cell types residing in the mucosa, enteric neurons and glia are not generated within the gut. Instead, they arise from neural crest cells that migrate into and colonise the developing gastrointestinal tract. Although they are "later" arrivals into the developing gut, enteric neural crest-derived cells (ENCCs) respond to many of the same secreted signalling molecules as the "resident" epithelial and mesenchymal cells, and several factors that control the development of smooth muscle cells, interstitial cells and epithelial cells also regulate ENCCs. Much progress has been made towards understanding the migration of ENCCs along the gastrointestinal tract and their differentiation into neurons and glia. However, our understanding of how enteric neurons begin to communicate with each other and extend their neurites out of the developing plexus layers to innervate the various cell types lining the concentric layers of the gastrointestinal tract is only beginning. It is critical for postpartum survival that the gastrointestinal tract and its enteric circuitry are sufficiently mature to cope with the influx of nutrients and their absorption that occurs shortly after birth. Subsequently, colonisation of the gut by immune cells and microbiota during postnatal development has an important impact that determines the ultimate outline of the intrinsic neural networks of the gut. In this review, we describe the integrated development of the ENS and its target cells.

Motility changes induced by intraluminal FeSO4 in guinea pig jejunum.

BACKGROUND: Dietary iron supplementation is associated with gastrointestinal (GI) side effects including vomiting, nausea, and diarrhea. Although inorganic iron in high concentrations may be damaging to the intestinal mucosa, we hypothesize that there are physiological effects on the GI tract that occur at concentrations achieved by supplementation. Thus, our aim was to investigate the effect of intraluminal ferrous sulfate (FeSO4 ) on jejunal motility. METHODS: Segments of guinea pig jejunum were cannulated and the intraluminal pressure recorded with a transducer, while movements were recorded with a video camera. Peristaltic threshold was the oral pressure that evoked four consecutive propulsive contractions. The nutrients decanoic acid (1 mM), l-phenylalanine (50 mM), or the micronutrient FeSO4 (1 mM) were infused intraluminally. We also tested the effect of FeSO4 on electrochemically detected serotonin (5-HT, 5-hydroxytryptamine) released from in vitro tissues, both at rest and following mechanical stimulation. KEY RESULTS: The jejuna peristaltic threshold was significantly decreased by all three nutrients: FeSO4 : 31 ± 2-23 ± 3 mmH2 O; decanoic acid: 27 ± 2-14 ± 2 mmH2 O; and l-phenylalanine: 30 ± 3-14 ± 3mmH2 O. Of the three, only decanoic acid induced segmentation, while FeSO4 inhibited decanoic acid-induced segmentation. Resting 5-HT release was increased by FeSO4 (128% of control), but mechanically evoked 5-HT release was reduced (70% of control). CONCLUSIONS & INFERENCES: These data suggest that some luminal effects of inorganic iron on jejunal motility could be mediated through a pathway involving altered release of 5-HT. A better understanding of the interaction between luminal iron and 5-HT containing enterochromaffin cells could improve iron supplementation strategies, thus reducing side effects.

Endogenous peptide YY and neuropeptide Y inhibit colonic ion transport, contractility and transit differentially via Y1 and Y2 receptors.

BACKGROUND AND PURPOSE: Peptide YY (PYY) and neuropeptide Y (NPY) activate Y receptors, targets under consideration as treatments for diarrhoea and other intestinal disorders. We investigated the gastrointestinal consequences of selective PYY or NPY ablation on mucosal ion transport, smooth muscle activity and transit using wild-type, single and double peptide knockout mice, comparing mucosal responses with those from human colon. EXPERIMENTAL APPROACH: Mucosae were pretreated with a Y1 (BIBO3304) or Y2 (BIIE0246) receptor antagonist and changes in short-circuit current recorded. Colonic transit and colonic migrating motor complexes (CMMCs) were assessed in vitro and upper gastrointestinal and colonic transit measured in vivo. KEY RESULTS: Y receptor antagonists revealed tonic Y1 and Y2 receptor-mediated antisecretory effects in human and wild-type mouse colon mucosae. In both, Y1 tone was epithelial while Y2 tone was neuronal. Y1 tone was reduced 90% in PYY⁻/⁻ mucosa but unchanged in NPY⁻/⁻ tissue. Y2 tone was partially reduced in NPY⁻/⁻ or PYY⁻/⁻ mucosae and abolished in tetrodotoxin-pretreated PYY⁻/⁻ tissue. Y1 and Y2 tone were absent in NPYPYY⁻/⁻ tissue. Colonic transit was inhibited by Y1 blockade and increased by Y2 antagonism indicating tonic Y1 excitation and Y2 inhibition respectively. Upper GI transit was increased in PYY⁻/⁻ mice only. Y2 blockade reduced CMMC frequency in isolated mouse colon. CONCLUSIONS AND IMPLICATIONS: Endogenous PYY and NPY induced significant mucosal antisecretory tone mediated by Y1 and Y2 receptors, via similar mechanisms in human and mouse colon mucosa. Both peptides contributed to tonic Y2-receptor-mediated inhibition of colonic transit in vitro but only PYY attenuated upper GI transit.

Activation of neuronal SST1 and SST2 receptors decreases neurogenic secretion in the guinea-pig jejunum.

BACKGROUND: Vasoactive intestinal peptide (VIP) submucosal neurons, the main regulators of gut secretion, display inhibitory postsynaptic potentials mediated by somatostatin (SOM) acting on SST(1) and SST(2) receptors (SSTR(1), SSTR(2)) in the guinea-pig small intestine. We investigated the implications of this for neurally-evoked mucosal secretion. METHODS: Mucosal-submucosal preparations from guinea-pig jejunum were mounted in Ussing chambers to measure Cl(-) secretion, measured by short circuit current (I(sc)). All drugs were added serosally. Veratridine (1 µmol L(-1)) was used to stimulate neurons and provide a robust secretory response for pharmacological testing.5-hydroxytrptamine (5-HT, 300 nmol L(-1)) was used to specifically activate non-cholinergic secretomotor neurons, while 1,1-dimethyl-4-phenylpiperazinium (DMPP, 10 µmol L(-1)) was used to stimulate all secretomotor neurons. KEY RESULTS: Somatostatin (50 nmol L(-1)) induced a tetrodotoxin (TTX, 1 µmol L(-1))-sensitive decrease in secretion. Somatostatin also reduced the veratridine-induced increase in I(sc). The effects of SOM were significantly reduced by blocking SSTR(1) and SSTR(2) individually or together. Blocking SSTR(1) abolished the inhibition produced by SOM. Quantitative PCR demonstrated that SSTR(1) and SSTR(2) were much more highly expressed in the submucosa than the mucosa. Submucosal SSTR(1) expression was several fold higher than SSTR(2). Responses to DMPP (biphasic) and 5-HT (monophasic) were TTX-sensitive. Somatostatin significantly reduced the 5-HT-induced increase in I(sc), and the second, more sustained phase evoked by DMPP. CONCLUSIONS & INFERENCES: These data suggest that SOM exerts its antisecretory effects by suppressing firing of VIP secretomotor neurons, rather than via a direct action on mucosal enterocytes.

The neurochemistry and innervation patterns of extrinsic sensory and sympathetic nerves in the myenteric plexus of the C57Bl6 mouse jejunum.

In vitro anterograde tracing of axons in mesenteric nerve trunks using biotinamide in combination with immunohistochemical labelling was used to characterize the extrinsic nerve projections in the myenteric plexus of the mouse jejunum. Anterogradely-labelled spinal sensory fibres innervating the enteric nervous system were identified by their immunoreactivity for calcitonin gene-related peptide (CGRP), while sympathetic noradrenergic fibres were detected with tyrosine hydroxylase (TH), using confocal microscopy. The presence of these markers has been previously described in the spinal sensory and sympathetic fibres. Labelled extrinsic nerve fibres in the myenteric plexus were identified apposing enteric neurons that were immunoreactive for either calretinin (CalR), calbindin (CalB) or nitric oxide synthase (NOS). Of the total anterogradely labelled axons in the myenteric plexus, 20% were CGRP-immunoreactive. Labelled CGRP-immunoreactive varicosities were closely apposed to CalR-immunoreactive myenteric cells, many of which were Dogiel type I (40%; interneurons) or type II (20%; intrinsic sensory) neurons. Labelled CGRP-immunoreactive varicosities were also observed in close appositions to CalB-immunoreactive myenteric cell bodies, of which a small subset had type II morphology (18%; intrinsic sensory neurons). A further 43% of all biotinamide-filled fibres were immunoreactive for TH and these fibres were apposed to CalR-immunoreactive cell bodies (small-sized; excitatory motor neurons) and NOS-immunoreactive cell bodies (either type I or small neurons; inhibitory motor neurons and interneurons) in the myenteric plexus. The results provide a neurochemical and neuroanatomical basis for connections between dorsal root afferent neurons and myenteric neurons and suggest an anatomical substrate for the well-known modulation of enteric circuits from sympathetic nerves. No anterogradely-labelled fibres were stained for NOS-immunoreactivity, despite more than 60% of dorsal root ganglion (DRG) neurons retrogradely labelled from the jejunum showing NOS-immunoreactivity. This was due to a substantial, time-dependent, and apparently selective, loss of NOS from extrinsic axons under in vitro conditions. Lastly, a small population of non-immunoreactive biotinamide-filled fibres (<1%) gave rise to dense terminal structures around individual myenteric cell bodies lacking CalR, CalB or NOS. These specialized endings may represent vagal fibres or a subset of spinal sensory neurons that do not contain CGRP.

Neurochemical and morphological phenotypes of vagal afferent neurons innervating the adult mouse jejunum.

Whilst much is known about the function and influence of vagal afferents on the mammalian upper gastrointestinal tract, the phenotypes of the different types of vagal afferent neurons innervating the jejunum is unknown. We have previously shown that spinal afferents supplying the jejunum are predominantly medium-sized sensory neurons that express specific combinations of transient receptor potential vanilloid type 1 (TRPV1), neuronal nitric oxide synthase (NOS) and calcitonin-gene related peptide (CGRP) and that they lack binding for isolectin B4 (IB4). This study aimed to identify the chemical phenotypes and somal sizes of jejunal afferent neurons in the mouse vagal ganglion. Jejunal vagal afferents were identified by retrograde labelling with sub-serosal injections of cholera toxin B (CTB) into the jejunal wall and assessed for IB4-binding, TRPV1-, NOS- and CGRP-immunoreactivities using fluorescent microscopy. Almost all (99%) of CTB-labelled vagal afferent neurons were small- and medium-sized sensory cells. Most (81%) jejunal vagal afferents bound IB4 but fewer (32%) expressed TRPV1. A quarter (25%) of those that bound IB4 co-expressed TRPV1-immunoreactivity whilst 77% of TRPV1-expressing jejunal vagal afferent neurons bound IB4. NOS (0%) and CGRP (0%) expression was absent from all CTB-labelled cells examined. In conclusion, vagal afferents innervating the jejunum differ in their expression of IB4, TRPV1, CGRP and NOS from their spinal counterparts, suggesting that the peripheral endings for extrinsic sensory neurons terminating within the enteric nervous system can be identified selectively.

Indirect evidence for increased mechanosensitivity of jejunal secretomotor neurones in patients with idiopathic bile acid malabsorption.

AIM: The interdigestive motor rhythm, the migrating motor complex (MMC), is accompanied by active secretion of chloride during periods of distally propagating maximal motor activity (MMC phase III). We studied the behaviour of this system in bile acid malabsorption (BAM), a relative common cause of chronic diarrhoea. We measured motor activity and transmucosal potential difference (PD, reflecting active chloride secretion), in the proximal jejunum in healthy controls (n = 18) and in a group of patients with BAM (n = 11). The phase III-generated voltage was related to the degree of BAM quantified by the (75)SeHCAT test. METHODS: We used a multi-channel intestinal infusion system to simultaneously measure jejunal pressure and PD. Saline passing calomel half-cells was infused into the jejunum and subcutaneously. Pressure and PD were recorded in the fasting state and after a test meal. RESULTS: In the absence of motor activity, jejunal PD was not significantly different from zero in either group. During MMC phase III, PD reached significantly higher mean and peak levels in BAM patients. The product of MMC phase III length multiplied by voltage, over 3 h, was also significantly higher in BAM patients (controls: median 307 mV x cm, range 70-398; BAM: median 511, range 274-2271, P < 0.01). This value was also significantly correlated with the degree of BAM as reflected by the (75)SeHCAT test (P < 0.05). CONCLUSION: Phase III induced jejunal secretion may be upregulated in BAM patients, resulting in overload of colonic reabsorption capacity.

Distinct chemical classes of medium-sized transient receptor potential channel vanilloid 1-immunoreactive dorsal root ganglion neurons innervate the adult mouse jejunum and colon.

Physiological studies suggest visceral spinal afferents are generally small diameter, unmyelinated C-fibers or myelinated Adelta-fibers, but little is known about the size and chemical phenotypes of visceral sensory neurons supplying the small intestine. This study examines the size and expression patterns of transient receptor potential vanilloid 1 (TRPV1), calcitonin gene-related peptide (CGRP), substance P (SP), neuronal nitric oxide synthase (NOS) and isolectin B4-binding (IB4) in dorsal root ganglion (DRG) neurons projecting to the gastrointestinal tract. The spinal afferent innervation of mouse jejunum and distal colon was investigated with retrograde neuronal tracing and multi-label immunohistochemistry. Expression of histochemical markers and soma sizes of retrogradely labeled DRG profiles were determined with confocal microscopy. Most (>75%) jejunal and colonic afferent neurons were medium- and large-sized cells. The majority (82%) of jejunal afferents expressed TRPV1, but few bound IB4. All retrogradely labeled jejunal afferents expressing NOS-immunoreactivity (64%) also expressed TRPV1 and CGRP and most expressed SP. Most labeled colonic afferents expressed TRPV1 (62%) and half expressed NOS. Taken together these data demonstrate that the spinal afferent supply of the jejunum and colon is largely from medium and large sensory neurons, suggesting most intestinal afferent axons are A fibers. The various chemically-defined subpopulations of afferents may play multiple roles in sensory innervation of the jejunum apart from nociceptive transduction. Additionally, we have identified a unique chemical code, TRPV1/NOS/CGRP/SP, that distinguishes many spinal afferent terminals from those of enteric neurons.

Synaptic transmission from the submucosal plexus to the myenteric plexus in Guinea-pig ileum.

Stimulation of the myenteric plexus results in activation of submucosal neurons and dilation of arterioles, one way that motility and secretion can be coupled together. The present study aimed to examine the converse, whether myenteric neurons receive synaptic input from the submucosal plexus (SMP). Intracellular recordings were made from guinea-pig ileal myenteric neurons while the SMP was electrically stimulated. Of the 29 neurons studied (13 S and 16 AH neurons), stimulation of the SMP evoked a synaptic potential in only seven cells, or 24% of neurons. When the SMP was situated oral to the myenteric plexus, 4 of 13 (31%) myenteric neurons had synaptic input. When it was situated circumferential, 2 of 8 (25%) had input, and when the SMP was situated anal 1 of 8 (13%) had input. Overall, 5 of the 13 (38%) S neurons responded with fast excitatory post-synaptic potentials (EPSPs), one of which also showed a slow EPSP, while 2 of the 16 (13%) AH neurons responded with a slow EPSP. This study indicates that the synaptic input from the SMP to myenteric neurons is relatively sparse. Whether this input is less important than the myenteric to submucosal input or simply represents a more selective form of control is unknown.

Purinergic mechanisms in the control of gastrointestinal motility.

For many years, ATP and adenosine have been implicated in movement regulation of the gastrointestinal tract. They act through three major receptor subtypes: adenosine or P1 receptors, P2X receptors and P2Y receptors. Each of these major receptor types can be subdivided into several different classes and is widely distributed amongst various neurons, muscle types, glia and interstitial cells that regulate intestinal functions. Several key roles for the different receptors and their endogenous ligands have been identified in physiological and pharmacological studies. For example, adenosine acting at A(1) receptors appears to inhibit intestinal motility in various pathological conditions. Similarly, ATP acting at P2Y receptors is an important component of inhibitory neuromuscular transmission, acting as a cotransmitter with nitric oxide. ATP acting at P2X and P2Y(1) receptors is important for synaptic transmission in simple descending excitatory and inhibitory reflex pathways. Some P2Y receptor subtypes prefer uridine nucleotides over purine nucleotides. Thus, roles for UTP and UDP as enteric transmitters in place of ATP cannot be excluded. ATP also appears to be important for sensory transduction, especially in chemosensitive pathways that initiate local inhibitory reflexes. Despite this evidence, data are lacking about the roles of either adenosine or ATP in more complex motility patterns such as segmentation or the interdigestive migrating motor complex. Clarification of roles for purinergic transmission in these common, but understudied, motility patterns will depend on the use of subtype-specific antagonists that in some cases have not yet been developed.

Targets of myenteric interneurons in the guinea-pig small intestine.

Polarized outputs of myenteric interneurons in guinea-pig small intestine have been well studied. However, the variety of motility patterns exhibited suggests that some interneuron targets remain unknown. We used antisera selected to distinguish interneuron varicosities and known myenteric neuron types to investigate outputs of three interneuron classes in guinea-pig jejunum; two classes of descending interneurons immunoreactive (IR) for somatostatin (SOM) or nitric oxide synthase (NOS)/vasoactive intestinal peptide (VIP), and one class of ascending interneurons [calretinin/enkephalin (ENK)-IR]. Varicosities apposed to immunohistochemically identified cell bodies were quantified by confocal microscopy. Intrinsic sensory neurons (calbindin-IR) were apposed by few varicosities. Cholinergic secretomotor neurons (neuropeptide Y-IR) were apposed by many SOM-IR varicosities. Longitudinal muscle excitatory motor neurons (calretinin-IR) were apposed by some VIP- and ENK-IR varicosities, but few SOM-IR varicosities. Ascending interneurons (calretinin-IR) were apposed by many varicosities of all types. NOS-IR interneurons and inhibitory motor neurons were apposed by numerous VIP-IR and SOM-IR varicosities. NOS-IR short inhibitory motor neurons were apposed by significantly fewer ENK-IR varicosities than other NOS-IR neurons. Based on the specific chemical coding of ascending (ENK) and descending (SOM) interneurons, we conclude that cholinergic secretomotor neurons and short inhibitory neurons are located in descending reflex pathways, while ascending interneurons and NOS-IR descending interneurons are focal points at which ascending and descending pathways converge.

Elevated motility-related transmucosal potential difference in the upper small intestine in the irritable bowel syndrome.

The pathophysiology of irritable bowel syndrome (IBS) is complex and incompletely known. Very little has been studied regarding the role of submucous neuronal activity. We therefore measured small intestinal transmural potential difference (PD, reflecting mainly electrogenic chloride secretion), and its linkage with fasting motor activity [migrating motor complex (MMC)] in controls (n = 16) and patients with IBS [n = 23, 14 diarrhoea predominant (d-IBS) and nine constipation predominant (c-IBS)]. Transmural-PD and its relation to MMC phase III was measured by modified multilumen manometry for 3 h in the fasting state using one jejunal and one duodenal infusion line as flowing electrodes. The amplitude and duration of motor phase III was similar in controls and IBS patients, but the propagation speed of phase III was higher in IBS patients. In IBS patients, maximal PD during MMC phase III was significantly elevated in both the duodenum and jejunum (P < 0.05) and the PD decline after phase III was significantly prolonged in the jejunum (P < 0.01). The PD elevation was seen in both duodenum and jejunum in d-IBS patients, but only in the jejunum in the c-IBS patients. On the basis of previous modelling studies, we propose that the enhanced secretion may reflect disturbed enteric network behaviour in some patients with IBS.

Local inhibitory reflexes excited by mucosal application of nutrient amino acids in guinea pig jejunum.

The motility of the gut depends on the chemicals contained in the lumen, but the stimuli that modify motility and their relationship to enteric neural pathways are unclear. This study examined local inhibitory reflexes activated by various chemical stimulants applied to the mucosa to characterize effective physiological stimuli and the pathways they excite. Segments of the jejunum were dissected to allow access to the circular muscle on one-half of the preparation while leaving the mucosa intact on the circumferentially adjacent half. Chemicals were transiently applied to the mucosa, and responses were recorded intracellularly in nearby circular muscle cells. The amino acids l-phenylalanine, l-alanine, or l-tryptophan (all 1 mM) evoked inhibitory junction potentials (IJPs; latency 150-300 ms, amplitude 3-8 mV, each n > 6) that were blocked by TTX and partially blocked by antagonists of P2X receptors and/or a combination of antagonists at 5-HT(3) and 5-HT(4) receptors. The putative mediators 5-HT (10 microM), ATP (1 mM), and CCK-8 (1-10 microM) elicited IJPs mediated via 5-HT(3), P2X, and CCK-B receptors, respectively. Responses were only partially reduced by the effective antagonists. IJPs evoked by electrically stimulating the mucosa were unaffected by antagonists that reduced chemically evoked responses. Both chemically and electrically evoked IJPs were resistant to nicotinic, NK(1), NK(3), alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid, N-methyl-d-aspartate, or CGRP receptor blockade. We conclude that mucosal stimulation by amino acids activates local neural pathways whose pharmacology depends on the nature of the stimulus. Transmitters involved at some synapses in these pathways remain to be identified.

Mechanisms underlying nutrient-induced segmentation in isolated guinea pig small intestine.

Mechanisms underlying nutrient-induced segmentation within the gut are not well understood. We have shown that decanoic acid and some amino acids induce neurally dependent segmentation in guinea pig small intestine in vitro. This study examined the neural mechanisms underlying segmentation in the circular muscle and whether the timing of segmentation contractions also depends on slow waves. Decanoic acid (1 mM) was infused into the lumen of guinea pig duodenum and jejunum. Video imaging was used to monitor intestinal diameter as a function of both longitudinal position and time. Circular muscle electrical activity was recorded by using suction electrodes. Recordings from sites of segmenting contractions showed they are always associated with excitatory junction potentials leading to action potentials. Recordings from sites oral and anal to segmenting contractions revealed inhibitory junction potentials that were time locked to those contractions. Slow waves were never observed underlying segmenting contractions. In paralyzed preparations, intracellular recording revealed that slow-wave frequency was highly consistent at 19.5 (SD 1.4) cycles per minute (c/min) in duodenum and 16.6 (SD 1.1) c/min in jejunum. By contrast, the frequencies of segmenting contractions varied widely (duodenum: 3.6-28.8 c/min, median 10.8 c/min; jejunum: 3.0-27.0 c/min, median 7.8 c/min) and sometimes exceeded slow-wave frequencies for that region. Thus nutrient-induced segmentation contractions in guinea pig small intestine do not depend on slow-wave activity. Rather they result from a neural circuit producing rhythmic localized activity in excitatory motor neurons, while simultaneously activating surrounding inhibitory motor neurons.

Mapping 5-HT inputs to enteric neurons of the guinea-pig small intestine.

5-HT released by gastrointestinal mucosa and enteric interneurons has powerful effects on gut behavior. However, the targets of 5-HT-containing neurons within enteric circuits are not well characterized. We used antisera against 5-HT and selected markers of known enteric neuron types to investigate the connections made by 5-HT-containing neurons in the guinea-pig jejunum. Confocal microscopy was used to quantify the number of 5-HT-immunoreactive varicosities apposed to immunohistochemically identified cell bodies. Large numbers of varicosities were identified apposing cholinergic secretomotor neurons, immunoreactive for neuropeptide Y, in both myenteric and submucous plexuses. Subgroups of neurons identified by calretinin (ascending interneurons) and nitric oxide synthase (descending interneurons and inhibitory motor neurons) immunoreactivity were also apposed by many varicosities. Longitudinal muscle motor neurons (calretinin immunoreactive) and AH/Dogiel type II (sensory) neurons (calbindin immunoreactive) were apposed by small numbers of varicosities. Combined retrograde tracing and immunohistochemistry were used to identify excitatory circular muscle motor neurons; these were encircled by 5-HT-immunoreactive varicosities, but the appositions could not be quantified. We suggest that 5-HT-containing interneurons are involved in secretomotor pathways and pathways to subgroups of other interneurons, but not longitudinal muscle motor neurons. There also appear to be connections between 5-HT-containing interneurons and excitatory circular muscle motor neurons. Physiological evidence demonstrates a functional connection between 5-HT-containing interneurons and AH/Dogiel type II neurons, but few 5-HT-immunoreactive varicosities were observed apposing calbindin-immunoreactive cell bodies. Taken together these results suggest that neural 5-HT may have significant roles in excitatory pathways regulating both motility and secretion.

Synaptic transmission at functionally identified synapses in the enteric nervous system: roles for both ionotropic and metabotropic receptors.

Digestion and absorption of nutrients and the secretion and reabsorption of fluid in the gastrointestinal tract are regulated by neurons of the enteric nervous system (ENS), the extensive peripheral nerve network contained within the intestinal wall. The ENS is an important physiological model for the study of neural networks since it is both complex and accessible. At least 20 different neurochemically and functionally distinct classes of enteric neurons have been identified in the guinea pig ileum. These neurons express a wide range of ionotropic and metabotropic receptors. Synaptic potentials mediated by ionotropic receptors such as the nicotinic acetylcholine receptor, P2X purinoceptors and 5-HT(3) receptors are seen in many enteric neurons. However, prominent synaptic potentials mediated by metabotropic receptors, like the P2Y(1) receptor and the NK(1) receptor, are also seen in these neurons. Studies of synaptic transmission between the different neuron classes within the enteric neural pathways have shown that both ionotropic and metabotropic synaptic potentials play major roles at distinct synapses within simple reflex pathways. However, there are still functional synapses at which no known transmitter or receptor has been identified. This review describes the identified roles for both ionotropic and metabotropic neurotransmission at functionally defined synapses within the guinea pig ileum ENS. It is concluded that metabotropic synaptic potentials act as primary transmitters at some synapses. It is suggested identification of the interactions between different synaptic potentials in the production of complex behaviours will require the use of well validated computer models of the enteric neural circuitry.

Different types of potassium channels underlie the long afterhyperpolarization in guinea-pig sympathetic and enteric neurons.

Ca(2+)-activated K(+) channels play an important role in the control of neuronal excitability via the generation of the afterhyperpolarization. While both small and large conductance Ca(2+)-activated K(+) channels underlie afterhyperpolarizations in different neuron types, the role of intermediate conductance Ca(2+)-activated K(+) channels (IK(Ca)) in the generation of afterhyperpolarizations remains unclear. The effects of blockade of IK(Ca) on guinea pig coeliac and ileal myenteric neurons were studied using single microelectrode current and voltage clamp. In coeliac neurons, TRAM-39, a selective blocker of IK(Ca), depressed the amplitude of the prolonged conductance underlying the slow afterhyperpolarization, (gKCa2) by 57%. In contrast, the conductance underlying the prolonged afterhyperpolarization in AH-type myenteric neurons was unaffected by TRAM-39, although it has been suggested that this AHP is mediated by IK(Ca). In both types of neurons, TRAM-39 did not alter the resting cell properties or the properties of the action potential. TRAM-39 had no effect on the amplitude of the fast component of the afterhyperpolarization present in sympathetic LAH neurons. The results of this study suggest that in sympathetic LAH neurons, activation of IK(Ca) underlies at least part of the prolonged afterhyperpolarization while the nature of the channel underlying the AHP in enteric neurons remains unclear.