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Neurons that originate from pre-vertebral sympathetic ganglia, the splanchnic-celiac-superior mesenteric ganglion complex (SCSMG) in mouse, have important roles in control of organs of the upper abdomen. Here, we present a protocol for the isolation of the mouse sympathetic SCSMG. We describe steps for surgical incision, ganglia isolation, ganglia fine dissection, and whole-mount SCSMG after clearing-enhanced 3D (Ce3D) clearing method and immunohistochemistry. Given the importance of mice in studies of that control, this protocol aims to assist biomedical researchers in the dissection of the mouse SCSMG., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

To investigate noxious stimulation-responsive neural circuits that could influence the gut, we recorded from intestinally directed (efferent) nerve filaments dissected from mesenteric nerves close to the small intestine in anesthetized rats. These exhibited baseline multiunit activity that was almost unaffected by vagotomy (VagX) and reduced only slightly by cutting the splanchnic nerves. The activity was halved by hexamethonium (Hex) treatment. When an adjacent gut segment received an intraluminal stimulus 2,4,6-trinitrobenzenesulfonate (TNBS) in 30% ethanol, mesenteric efferent nerve activity increased for more than 1 h. The increased activity was almost unaffected by bilateral vagotomy or splanchnic nerve section, indicating a lack of central nervous involvement, but it was 60% reduced by hexamethonium. Spike sorting discriminated efferent single and predominantly single-unit spike trains that responded to TNBS, were unaffected by splachnectomy but were silenced by hexamethonium. After noxious stimulation of one segment, the adjacent segment showed no evidence of suppression of gut motility or vasoconstriction. We conclude that luminal application of a noxious stimulus to the small intestine activates an entirely peripheral, intestinointestinal reflex pathway. This pathway involves enteric intestinofugal neurons that excite postganglionic sympathetic neurons via a nicotinic synapse. We suggest that the final sympathetic efferent neurons that respond to a tissue damaging stimulus are distinct from vasoconstrictor, secretomotor, and motility inhibiting neurons. NEW & NOTEWORTHY An intraluminal noxious chemical stimulus applied to one segment of small intestine increased mesenteric efferent nerve activity to an adjacent segment. This was identified as a peripheral ganglionic reflex that did not require vagal or spinal connections. Hexamethonium blocked most, but not all, ongoing and reflex mesenteric efferent activity. The prevertebral sympathetic efferent neurons that are activated likely affect inflammatory and immune functions of other gut segments.

The stomach is the primary reservoir of the gastrointestinal tract, where ingested content is broken down into small particles. Coordinated relaxation and contraction is essential for rhythmic motility and digestion, but how the muscle motor innervation is organized to provide appropriate graded regional control is not established. In this study, we recorded neuromuscular transmission to the circular muscle using intracellular microelectrodes to investigate the spread of the influence of intrinsic motor neurons. In addition, microanatomical investigations of neuronal projections and pharmacological analysis were conducted to investigate neuromuscular relationships. We found that inhibitory neurotransmission to the circular muscle is graded with stimulus strength and circumferential distance from the stimulation site. The influence of inhibitory neurons declined between 1 and 11 mm from the stimulation site. In the antrum, corpus, and fundus, the declines at 11 mm were about 20%, 30%, and 50%, respectively. Stimulation of inhibitory neurons elicited biphasic hyperpolarizing potentials often followed by prolonged depolarizing events in the distal stomach, but only hyperpolarizing events in the proximal stomach. Excitatory neurotransmission influence varied greatly between proximal stomach, where depolarizing events occurred, and distal stomach, where no direct electrical effects in the muscle were observed. Structural studies using microlesion surgeries confirmed a dominant circumferential projection. We conclude that motor neuron influences extend around the gastric circumference, that the effectiveness can be graded by the recruitment of different numbers of motor neuron nerve terminals to finely control gastric motility, and that the ways in which the neurons influence the muscle differ between anatomical regions. NEW & NOTEWORTHY This study provides a detailed mapping of nerve transmission to the circular muscle of the different anatomical regions of rat stomach. It shows that excitatory and inhibitory influences extend around the gastric circumference and that there is a summation of neural influence that allows for finely graded control of muscle tension and length. Nerve-mediated electrical events are qualitatively and quantitatively different between regions, for example, excitatory neurons have direct effects on fundus but not antral muscle.

We investigated the distributions and targets of nitrergic neurons in the rat stomach, using neuronal nitric oxide synthase (NOS) immunohistochemistry and nicotinamide adenine dinucleotide phosphate (NADPH) diaphorase histochemistry. Nitrergic neurons comprised similar proportions of myenteric neurons, about 30%, in all gastric regions. Small numbers of nitrergic neurons occurred in submucosal ganglia. In total, there were ~ 125,000 neuronal nitric oxide synthase (nNOS) neurons in the stomach. The myenteric cell bodies had single axons, type I morphology and a wide range of sizes. Five targets were identified, the longitudinal, circular and oblique layers of the external muscle, the muscularis mucosae and arteries within the gastric wall. The circular and oblique muscle layers had nitrergic fibres throughout their thickness, while the longitudinal muscle was innervated at its inner surface by fibres of the tertiary plexus, a component of the myenteric plexus. There was a very dense innervation of the pyloric sphincter, adjacent to the duodenum. The muscle strands that run between mucosal glands rarely had closely associated nNOS nerve fibres. Both nNOS immunohistochemistry and NADPH histochemistry showed that nitrergic terminals did not provide baskets of terminals around myenteric neurons. Thus, the nitrergic neuron populations in the stomach supply the muscle layers and intramural arteries, but, unlike in the intestine, gastric interneurons do not express nNOS. The large numbers of nNOS neurons and the density of innervation of the circular muscle and pyloric sphincter suggest that there is a finely graded control of motor function in the stomach by the recruitment of different numbers of inhibitory motor neurons., (© 2022. The Author(s).)

The strengths, directions and coupling of the movements of the stomach depend on the organisation of its musculature. Although the rat has been used as a model species to study gastric function, there is no detailed, quantitative study of the arrangement of the gastric muscles in rat. Here we provide a descriptive and quantitative account, and compare it with human gastric anatomy. The rat stomach has three components of the muscularis externa, a longitudinal coat, a circular coat and an internal oblique (sling) muscle in the region of the gastro-oesophageal junction. These layers are similar to human. Unlike human, the rat stomach is also equipped with paired muscular oesophago-pyloric ligaments that lie external to the longitudinal muscle. There is a prominent muscularis mucosae throughout the stomach and strands of smooth muscle occur in the mucosa, between the glands of the corpus and antrum. The striated muscle of the oesophageal wall reaches to the stomach, unlike the human, in which the wall of the distal oesophagus is smooth muscle. Thus, the continuity of gastric and oesophageal smooth muscle bundles, that occurs in human, does not occur in rat. Circular muscle bundles extend around the circumference of the stomach, in the fundus forming a cap of parallel muscle bundles. This arrangement favours co-ordinated circumferential contractions. Small bands of muscle make connections between the circular muscle bundles. This is consistent with a slower conduction of excitation orthogonal to the circular muscle bundles, across the corpus towards the distal antrum. The oblique muscle merged and became continuous with the circular muscle close to the gastro-oesophageal junction at the base of the fundus, and in the corpus, lateral to the lesser curvature. Quantitation of muscle thickness revealed gradients of thickness of both the longitudinal and circular muscle. This anatomical study provides essential data for interpreting gastric movements., (© 2021 Anatomical Society.)