Melina Ellis

The first intestinal motility patterns in fetal mice are not mediated by neurons or interstitial cells of Cajal

In mature animals, neurons and interstitial cells of Cajal (ICC) are essential for organized intestinal motility. We investigated motility patterns, and the roles of neurons and myenteric ICC (ICC‐MP), in the duodenum and colon of developing mice in vitro. Spatiotemporal mapping revealed regular contractions that propagated in both directions from embryonic day (E)13.5 in the duodenum and E14.5 in the colon. The propagating contractions, which we termed ripples, were unaffected by tetrodotoxin and were present in the intestine of embryonic Ret null mutant mice, which lack enteric neurons. Neurally mediated motility patterns were first observed in the duodenum at E18.5. To examine the possible role of ICC‐MP, three approaches were used. First, intracellular recordings from the circular muscle of the duodenum did not detect slow wave activity at E16.5, but regular slow waves were observed in some preparations of E18.5 duodenum. Second, spatiotemporal mapping revealed ripples in the duodenum of E13.5 and E16.5 W/Wv embryos, which lack KIT+ ICC‐MP and slow waves. Third, KIT‐immunoreactive cells with the morphology of ICC‐MP were first observed at E18.5. Hence, ripples do not appear to be mediated by ICC‐MP and must be myogenic. Ripples in the duodenum and colon were abolished by cobalt chloride (1 mm). The L‐type Ca2+ channel antagonist nicardipine (2.5 μm) abolished ripples in the duodenum and reduced their frequency and size in the colon. Our findings demonstrate that prominent propagating contractions (ripples) are present in the duodenum and colon of fetal mice. Ripples are not mediated by neurons or ICC‐MP, but entry of extracellular Ca2+ through L‐type Ca2+ channels is essential. Thus, during development of the intestine, the first motor patterns to develop are myogenic.

Cholera Toxin Induces Sustained Hyperexcitability in Submucosal Secretomotor Neurons in Guinea Pig Jejunum

BACKGROUND & AIMS Neural mechanisms underlying cholera toxin (CT)-induced intestinal hypersecretion remain unclear. We investigated long-term excitability changes in vasoactive intestinal peptide (VIP) and neuropeptide Y (NPY) secretomotor neurons after prolonged luminal exposure to CT. METHODS Isolated segments of guinea pig jejunum were incubated with saline or CT +/- neurotransmitter antagonist in the lumen; the submucosal plexus was then dissected clear, circumferentially adjacent to intact mucosa. Synaptic inputs and firing properties of S neurons in ganglia next to the mucosa in control saline were studied using intracellular recording. Neurons were processed for VIP and NPY immunoreactivity. RESULTS Thirty S neurons (20 VIP(+), 7 NPY(+), 3 VIP(-)/NPY(-)) from CT-treated preparations and 27 control S neurons (19 VIP(+), 4 NPY(+), 4 VIP(-)/NPY(-)) in ganglia adjacent to intact mucosa were analyzed. VIP(+) and NPY(+) neurons in CT-treated preparations fired significantly more action potentials and for longer periods during injected depolarizing current pulses (50-350 pA) than control neurons. Addition of tetrodotoxin, hexamethonium, granisetron, or the neurokinin-1 (NK1) antagonist SR140333 during the CT incubation blocked CT-induced effects in both neuron types. The NK3 antagonist SR142801 blocked CT-induced effects in NPY(+) neurons and reduced the number of action potentials in VIP(+) neurons. Synaptic activity was unaffected by CT. CONCLUSIONS CT induces specific and sustained hyperexcitability of secretomotor neurons in enteric pathways. CT acts in the mucosa. Its effect is neurally mediated and depends on 5-hydroxytryptamine-3, nicotinic, and NK1 receptors. This system represents a unique model to understand the neural mechanisms of action of CT and to identify therapeutic targets.

Luminal Cholera Toxin Alters Motility in Isolated Guinea-Pig Jejunum via a Pathway Independent of 5-HT3 Receptors

Cholera toxin (CT) is well established to produce diarrhea by producing hyperactivity of the enteric neural circuits that regulate water and electrolyte secretion. Its effects on intestinal motor patterns are less well understood. We examined the effects of luminal CT on motor activity of guinea-pig jejunum in vitro. Segments of jejunum were cannulated at either end and mounted horizontally. Their contractile activity was video-imaged and the recordings were used to construct spatiotemporal maps of contractile activity with CT (1.25 or 12.5 μg/ml) in the lumen. Both concentrations of CT induced propulsive motor activity in jejunal segments. The effect of 1.25 μg/ml CT was markedly enhanced by co-incubation with granisetron (5-HT3 antagonist, 1 μM), which prevents the hypersecretion induced by CT. The increased propulsive activity was not accompanied by increased segmentation and occurred very early after exposure to CT in the presence of granisetron. Luminal CT also reduced the pressure threshold for saline distension evoked propulsive reflexes, an effect resistant to granisetron. In contrast, CT prevented the induction of segmenting contractions by luminal decanoic acid, so its effects on propulsive and segmenting contractile activity are distinctly different. Thus, in addition to producing hypersecretion, CT excites propulsive motor activity with an entirely different time course and pharmacology, but inhibits nutrient-induced segmentation. This suggests that CT excites more than one enteric neural circuit and that propulsive and segmenting motor patterns are differentially regulated.

Video Imaging and Spatiotemporal Maps to Analyze Gastrointestinal Motility in Mice.

The enteric nervous system (ENS) plays an important role in regulating gastrointestinal (GI) motility and can function independently of the central nervous system. Changes in ENS function are a major cause of GI symptoms and disease and may contribute to GI symptoms reported in neuropsychiatric disorders including autism. It is well established that isolated colon segments generate spontaneous, rhythmic contractions known as Colonic Migrating Motor Complexes (CMMCs). A procedure to analyze the enteric neural regulation of CMMCs in ex vivo preparations of mouse colon is described. The colon is dissected from the animal and flushed to remove fecal content prior to being cannulated in an organ bath. Data is acquired via a video camera positioned above the organ bath and converted to high-resolution spatiotemporal maps via an in-house software package. Using this technique, baseline contractile patterns and pharmacological effects on ENS function in colon segments can be compared over 3-4 hr. In addition, propagation length and speed of CMMCs can be recorded as well as changes in gut diameter and contraction frequency. This technique is useful for characterizing gastrointestinal motility patterns in transgenic mouse models (and in other species including rat and guinea pig). In this way, pharmacologically induced changes in CMMCs are recorded in wild type mice and in the Neuroligin-3(R451C) mouse model of autism. Furthermore, this technique can be applied to other regions of the GI tract including the duodenum, jejunum and ileum and at different developmental ages in mice.