Stimulation of extrinsic sympathetic nerves differentially affects neurogenic motor activity in guinea pig distal colon.

The speed of pellet propulsion through the isolated guinea pig distal colon in vitro significantly exceeds in vivo measurements, suggesting a role for inhibitory mechanisms from sources outside the gut. The aim of this study was to investigate the effects of sympathetic nerve stimulation on three different neurogenic motor behaviors of the distal colon: transient neural events (TNEs), colonic motor complexes (CMCs), and pellet propulsion. To do this, segments of guinea pig distal colon with intact connections to the inferior mesenteric ganglion (IMG) were set up in organ baths allowing for simultaneous extracellular suction electrode recordings from smooth muscle, video recordings for diameter mapping, and intraluminal manometry. Electrical stimulation (1-20 Hz) of colonic nerves surrounding the inferior mesenteric artery caused a statistically significant, frequency-dependent inhibition of TNEs, as well as single pellet propulsion, from frequencies of 5 Hz and greater. Significant inhibition of CMCs required stimulation frequencies of 10 Hz and greater. Phentolamine (3.6 µM) abolished effects of colonic nerve stimulation, consistent with a sympathetic noradrenergic mechanism. Sympathetic inhibition was constrained to regions with intact extrinsic nerve pathways, allowing normal motor behaviors to continue without modulation in adjacent extrinsically denervated regions of the same colonic segments. The results demonstrate differential sensitivities to sympathetic input among distinct neurogenic motor behaviors of the colon. Together with findings indicating CMCs activate colo-colonic sympathetic reflexes through the IMG, these results raise the possibility that CMCs may paradoxically facilitate suppression of pellet movement in vivo, through peripheral sympathetic reflex circuits.

Identifying Types of Neurons in the Human Colonic Enteric Nervous System.

Distinguishing and characterising the different classes of neurons that make up a neural circuit has been a long-term goal for many neuroscientists. The enteric nervous system is a large but moderately simple part of the nervous system. Enteric neurons in laboratory animals have been extensively characterised morphologically, electrophysiologically, by projections and immunohistochemically. However, studies of human enteric nervous system are less advanced despite the potential availability of tissue from elective surgery (with appropriate ethics permits). Recent studies using single cell sequencing have confirmed and extended the classification of enteric neurons in mice and human, but it is not clear whether an encompassing classification has been achieved. We present preliminary data on a means to distinguish classes of myenteric neurons in specimens of human colon combining immunohistochemical, morphological, projection and size data on single cells. A method to apply multiple layers of antisera to specimens was developed, allowing up to 12 markers to be characterised in individual neurons. Applied to multi-axonal Dogiel type II neurons, this approach demonstrated that they constitute fewer than 5% of myenteric neurons, are nearly all immunoreactive for choline acetyltransferase and tachykinins. Many express the calcium-binding proteins calbindin and calretinin and they are larger than average myenteric cells. This methodology provides a complementary approach to single-cell mRNA profiling to provide a comprehensive account of the types of myenteric neurons in the human colon.

Mechanisms underlying initiation of propulsion in guinea pig distal colon.

Colonic motor complexes (CMCs) are a major neurogenic activity in guineapig distal colon. The identity of the enteric neurons that initiate this activity is not established. Specialized intrinsic primary afferent neurons (IPANs) are a major candidate. We aimed to test this hypothesis. To do this, segments of guineapig distal colon were suspended vertically in heated organ baths and propulsive forces acting on a pellet inside the lumen were recorded by isometric force transducer while pharmacological agents were applied to affect IPAN function. In the absence of drugs, CMCs acted periodically on the pellet, generating peak propulsive forces of 12.7 ± 5 g at 0.56 ± 0.22 cpm, lasting 49 ± 17 s (215 preparations; n = 60). Most but not all CMCs were abolished by nicotinic receptor blockade to inhibit fast excitatory synaptic transmission (50/62 preparations; n = 25). Remarkably, CMCs inhibited by hexamethonium were restored by a pharmacological strategy that aimed to enhance IPAN excitability. Thus, CMCs were restored by increased smooth muscle tension (using BAY K8644, bethanechol or carbachol) and by IPAN excitation using phorbol dibutyrate; NK3 receptor agonist, senktide; and partially by αCGRP. The IPAN inhibitor, 5,6-dichloro-1-ethyl-1,3-dihydro-2H-benzimidazole-2-one (DCEBIO), decreased CMC frequency. CGRP, but not NK3-receptor antagonists, decreased CMC frequency in naive preparations. Finally, CMCs were blocked by tetrodotoxin, and this was not reversed by any drugs listed above. These results support a major role for IPANs that does not require fast synaptic transmission, in the periodic initiation of neurogenic propulsive contractions. Endogenous CGRP plays a role in determining CMC frequency, whereas further unidentified signaling pathways may determine their amplitude and duration.NEW & NOTEWORTHY The colonic motor complex (CMC) initiates propulsion in guinea pig colon. Here, CMCs evoked by an intraluminal pellet were restored during nicotinic receptor blockade by pharmacological agents that directly or indirectly enhance intrinsic primary afferent neuron (IPAN) excitability. IPANs are the only enteric neuron in colon that contain CGRP. Blocking CGRP receptors decreased CMC frequency, implicating their role in CMC initiation. The results support a role for IPANs in the initiation of CMCs.

The human enteric nervous system. Historical and modern advances. Collaboration between science and surgery.

BACKGROUND: There are considerable advantages and opportunities for surgeons and trainee surgeons in conducting a period of research allied with basic scientists. Such clinicians are well placed to define relevant clinical questions, provide human material (tissue, biopsy and blood) and translate the techniques derived in experimental animals to human subjects. METHODS: This small review explores research conducted on the nervous system of the intestines, with an emphasis on the translation of findings from animal to human. RESULTS: This work shows that new techniques of immunohistochemistry and retrograde tracing, developed in animal tissue, have greatly expanded our knowledge of the structure of the human enteric nervous system. CONCLUSIONS: Such findings have sparked therapeutic trials for the treatment of gastrointestinal disorders in patients.

Postoperative ileus-An ongoing conundrum.

BACKGROUND: Postoperative ileus is common and is a major clinical problem. It has been widely studied in patients and in experimental models in laboratory animals. A wide variety of treatments have been tested to prevent or modify the course of this disorder. PURPOSE: This review draws together information on animal studies of ileus with studies on human patients. It summarizes some of the conceptual advances made in understanding the mechanisms that underlie paralytic ileus. The treatments that have been tested in human subjects (both pharmacological and non-pharmacological) and their efficacy are summarized and graded consistent with current clinical guidelines. The review is not intended to provide a comprehensive overview of ileus, but rather a general understanding of the major clinical problems associated with it, how animal models have been useful to elucidate key mechanisms and, finally, some perspectives from both scientists and clinicians as to how we may move forward with this debilitating yet common condition.

Characterization of alternating neurogenic motor patterns in mouse colon.

BACKGROUND: Colonic motor complexes (CMCs) have been widely recorded in the large intestine of vertebrates. We have investigated whether in the smooth muscle, a single unified pattern of electrical activity, or different patterns of electrical activity give rise to the different neurogenic patterns of motility underlying CMCs in vitro. METHODS: To study differences of the CMCs between proximal and distal colon, we used a novel combination of techniques to simultaneously record muscle diameter and force at multiple sites along the whole mouse colon ex vivo. In addition, electrical activity of smooth muscle was recorded by suction electrodes. KEY RESULTS: Two distinct types of CMCs were distinguished; CMCs that propagated along the entire colon (complete CMC) and CMCs which were restricted to the proximal colon (incomplete CMC). The two types of CMC often occurred in the same preparations. Incomplete CMCs had longer bursts of smooth muscle action potentials than complete CMCs and propagated more slowly. Interestingly, both types of CMC were associated with similar frequency bursts of smooth muscle action potentials at ~2.4 Hz. In the most proximal colon, an additional firing frequency was detected close to ~7 Hz generating multiple peaks within each CMC. CONCLUSIONS & INFERENCES: We report distinct characteristics underlying complete and incomplete CMCs in isolated mouse colon. Recognizing these distinct patterns of motility will be important for future interpretation of analysis of murine colonic motility recordings. The identification of alternating patterns of motor activity in proximal colon, but not distal colon may reflect specific neural mechanisms for fecal pellet formation.

Intrinsic sensory neurons provide direct input to motor neurons and interneurons in mouse distal colon via varicose baskets.

Connections from intrinsic primary afferent neurons (IPANs), to ascending motor and interneurons have been described in guinea pig colon. These mono- and polysynaptic circuits may underlie polarized motor reflexes evoked by local gut stimulation. There is a need to translate findings in guinea pig to mouse, a species increasingly used in enteric neuroscience. Here, mouse distal colon was immunolabeled for CGRP, a marker of putative IPANs. This revealed a combination of large, intensely immunofluorescent axons in myenteric plexus and circular muscle, and thinner varicose axons with less immunofluorescence. The latter formed dense, basket-like varicosity clusters (CGRP+ baskets) that enveloped myenteric nerve cell bodies. Immunolabeling after 4-5 days in organ culture caused loss of large CGRP+ axons, but not varicose CGRP+ fibers and CGRP+ baskets. Baskets were characterized further by triple labeling with CGRP, nitric oxide synthase (NOS) and calretinin (CALR) antibodies. Approximately half (48%) of nerve cell bodies inside CGRP+ baskets lacked both NOS and CALR, while two overlapping populations containing NOS and/or CALR comprised the remainder. Quantitative analysis revealed CGRP+ varicosities were most abundant in baskets, followed by CALR+ varicosities, with a high degree of colocalization between the two markers. Few NOS+ varicosities occurred in baskets. Significantly higher proportions of CALR+ and CGRP+ varicosities colocalized in baskets than in circular muscle. In conclusion, CGRP+ baskets in mouse colon are formed by intrinsic enteric neurons with a neurochemical profile consistent with IPANs and have direct connections to both excitatory and inhibitory neurons.

Omental infarction mimicking cholecystitis.

Omental infarction can be difficult to diagnose preoperatively as imaging may be inconclusive and patients often present in a way that suggests a more common surgical pathology such as appendicitis. Here, a 40-year-old Caucasian man presented to casualty with shortness of breath and progressive right upper abdominal pain, accompanied with right shoulder and neck pain. Exploratory laparoscopy was eventually utilised to diagnose an atypical form of omental infarction that mimics cholecystitis. The vascular supply along the long axis of the segment was occluded initiating necrosis. In this case, the necrotic segment was adherent with the abdominal wall, a pathology not commonly reported in cases of omental infarction.

Transgene expression and transgene-induced silencing in diploid and autotetraploid Arabidopsis.

Previous studies have suggested that transgene expression in plants can be affected by ploidy. Here we show that three different transgenes, a reporter transgene, an antisense transgene, and a hairpin RNA (hpRNA) transgene, are all expressed at a lower level in autotetraploid (4n) than in diploid (2n) Arabidopsis. RNA silencing of two endogenous genes was induced by the antisense and hpRNA transgenes and this silencing is significantly less effective in 4n than in 2n Arabidopsis; furthermore, the reduced silencing in 4n Arabidopsis correlated with reduced accumulation of silencing-inducer RNAs. Methylation analysis both of independent 2n and 4n transgenic lines and of 2n and 4n progeny derived from the same 3n transgenic parent, indicated that transgenes are more methylated in 4n than 2n Arabidopsis. These results suggest that transgenes are transcriptionally repressed in the 4n background, resulting in expression levels lower than in the 2n background. Transgenes designed to silence endogenous genes express lower concentrations of silencing-inducer RNAs in 4n Arabidopsis plants, resulting in less effective silencing of target genes than in 2n Arabidopsis plants.