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.

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.

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.

Functional gastrointestinal disorders such as gastroparesis and achalasia can be caused by changes in the stomach’s intrinsic neural circuitry, the enteric nervous system. We used electrophysiological, immunohistochemistry and lesion techniques to map the function and structure of this circuitry, to aid in novel neuromodulation therapies to treat these debilitating disorders. It is expected that a single enteric neuron innervates multiple smooth muscle cells, and that an increased stimulus recruits more axons, evoking a summated response at the autonomic neuromuscular junction. Strips of muscle from different stomach regions of rats were taken for in vitro intracellular electrophysiology recordings from the circular muscle layer. Both inhibitory and excitatory neuromuscular junction potentials (JP) were measured. Inhibitory transmission was antagonised by a combination of nitric oxide synthase inhibitors and purine receptor antagonists and excitatory transmission was substantially reduced by muscarinic block. The relative amplitudes of excitatory and inhibitory JPs were quantified across all regions of the stomach, in most cases the relative amplitude was halved 9 millimetres (mm) circumferentially from the stimulation site. In the antrum, the whole circumference can be affected simultaneously. Innervation effectiveness at all distances was graded with stimulus strength. Suggesting each individual smooth muscle cell received multiple neuronal inputs. However, the relative amplitudes varied depending on the stomach region. Localisation studies show that the numbers of inhibitory and excitatory neurons are similar however the distribution of myenteric ganglia within the stomach were uneven from the lesser to greater curvature. This potentially influences the relative JP amplitudes recorded from different regions. Response amplitudes recorded closest to the lesser curvature were smaller when compared to the site further circumferentially. Furthermore, our results indicate that this may not be the only factor leading to the variabilities in innervation. Lesion and immunohistochemistry studies indicate that axons of inhibitory and excitatory neurons branch and extend circumferentially and the innervation fields of several neurons overlap. Our electrophysiology shows that the degree of influence of excitatory neurons on the smooth muscle cell varies dependent on the region recorded. We conclude that inhibitory and excitatory neural influences extend around the gastric circumference and the effectiveness of inhibitory and excitatory influences can be graded by the recruitment of different numbers of motor neuron nerve terminals. This study was supported by NIH (SPARC) grant, The Virtual Stomach (OT2OD030538), Principal Investigators Leo Cheng (University of Auckland) and Zhongming Liu (University of Michigan) This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

This protocol describes the methods used to evaluate neuronal target and population density in wholemount preparations and cryostat sections. In brief, stomachs from Sprague-Dawley rats were collected, fixed, cleared, and processed before being immunohistochemically stained with primary antibody and fluorescent secondary antibodies. Neuronal density and neuronal target analysis were completed using Zen Blue analysis software.

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.

Many central nervous system actions on final effectors such as the muscle and lining epithelium are exerted via the enteric nervous system. For example, the vagus nerves innervate enteric ganglia of the stomach, pancreas, and gallbladder, but not the muscle directly, sympathetic pathways innervate myenteric and submucosal ganglia, and pelvic nerves innervate the ganglia of the distal colon and rectum. However, some connections are direct, notably the vagal innervation of esophageal striated muscle, sympathetic innervation of the gastrointestinal sphincters, and, in large animals, muscle of nonsphincter regions and sympathetic innervation of gastrointestinal blood vessels. Immunohistochemical studies, combined with surgical denervation, confirm that both intrinsic cholinergic and noncholinergic neurons innervate submucosal arterioles. The structural organization of the circuits that detect the state of the small intestine, integrate the information and direct the activities of motor neurons is known and the colonic circuits appear to be similar although there are some specializations of colonic circuits.

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.

This protocol describes the methods used to quantify the surface area of the rat stomach. Scale photographs of rat stomachs, which were perfusion fixed, were used to determine the surface area of the three main regions of the rat stomach which enabled estimates of total neuronal populations to be calculated.

The functions of the stomach are powerfully influenced by the vagus nerve and vago-vagal reflexes. The vagal afferent neurons that innervate the stomach and monitor its functional state have clearly demonstrated roles in appetite regulation, gastric accommodation and gastric emptying. In mammals with single compartment stomachs, including humans, the stomach can be divided into functional regions and the muscle and mucosal layers show region-specific specializations. Electrophysiological and morphological studies suggest that the vagal sensory axons innervating the different regions and layers of the stomach wall have distinct properties and participate in different types of reflex controls. We therefore hypothesized that these functional and structural differences would be reflected in distinct gene expression profiles of afferent neurons, depending on the stomach region and tissue innervated. We developed methods to selectively retrogradely label afferent neurons innervating the muscle or mucosa in the corpus and antrum and the muscle in the fundus in the rat, using local microinjections of fluorescent nanospheres that diffuse minimally. Up to 30 microinjections of 30-100nL of tracer beads were made into either the muscle or the mucosa in one stomach region in each rat under anesthesia. The animal was allowed to recover and kept for 1-2 weeks before being humanely killed and tissues harvested. Labelled neurons were studied in situ in the nodose ganglion using confocal microscopy, immunohistochemistry and fluorescence in situ hybridization, and individual neurons collected after dissociation for RNAseq analysis. RNA sequencing was performed using pooled samples of 5-12 neurons labelled from each region and deep sequencing (30-50 million reads per sample), and then analyzed using minimal filtering to allow detection of genes expressed at low copy number. Nodose neurons labelled from the mucosa were significantly larger than those labelled from the muscle in both the corpus and antrum, whether quantified in situ or after dissociation, confirming that microinjections into the different stomach wall layers labelled different populations of neurons. Pairwise differential expression analyses of RNAseq data showed distinct mRNA expression profiles depending on the region innervated, and between muscle and mucosal innervating neurons in each region, based on Benjamini/Hochberg adjusted P values (Limma analysis tool). Some genes displayed region specific expression patterns while others showed tissue specific patterns (eg muscle vs mucosa) and some showed both. Among differentially expressed genes were some previously identified to specifically label gastric mucosal and muscle afferent neurons, such as somatostatin, CCKB receptor and GRP65. In addition, many other genes were differentially expressed and mRNA for genes known to be functional in vagal afferent neurons, such as ghrelin, leptin and galanin receptors, were detected. Differential expression patterns for various genes were confirmed in labelled neurons in situ using immunohistochemistry and/or in situhybridization. The data provide a rich resource for further studies of the visceral sensation in the different stomach regions and how region-specific signaling might regulate appetite and gastric function in both normal and disease states.

NOS (nitric oxide synthase) is a NADPH oxidase, requiring NADPH as a proton donor for the conversion of arginine to citrulline and the production of nitric oxide (NO). Thus nitrergic neurons can be localised by nNOS immunoreactivity or histochemically, using the NADPH diaphorase reaction (Young et al. 1992) to enable investigation of their morphology.

This review traces the history of the discovery and subsequent understanding of smooth muscle cells and their motor innervation. Smooth muscle tissue is made up of thousands of very small, individual, electrically connected, muscle cells. Each axon that enters a smooth muscle tissue branches extensively to form a terminal arbour that comes close to hundreds of smooth muscle cells. The branches of the terminal arbour are varicose, and each varicosity, of which there can be thousands, contains numerous transmitter storage vesicles. However, the probability of an individual varicosity releasing transmitter onto the adjacent muscle cells when an action potential passes is low. Many axons influence each muscle cell, some because they release transmitter close to the cell, and some because the events that they cause in other cells are electrically coupled to the cell under investigation. In tissues where this has been assessed, 20 or more axons can influence a single smooth muscle cell. We present a model of the innervation and influence of neurons on smooth muscle.

The gut immune system in the healthy intestine is anti-inflammatory, but can move to a pro-inflammatory state when the gut is challenged by pathogens or in disease. The nervous system influences the level of inflammation through enteric neurons and extrinsic neural connections, particularly vagal and sympathetic innervation of the gastrointestinal tract, each of which exerts anti-inflammatory effects. Within the enteric nervous system (ENS), three neuron types that influence gut immune cells have been identified, intrinsic primary afferent neurons (IPANs), vasoactive intestinal peptide (VIP) neurons that project to the mucosa, and cholinergic neurons that influence macrophages in the external muscle layers. The enteric neuropeptides, calcitonin gene-related peptide (CGRP), tachykinins, and neuromedin U (NMU), which are contained in IPANs, and VIP produced by the mucosa innervating neurons, all influence immune cells, notably innate lymphoid cells (ILCs). ILC2 are stimulated by VIP to release IL-22, which promotes microbial defense and tissue repair. Enteric neurons are innervated by the vagus, and, in the large intestine, by the pelvic nerves. Vagal nerve stimulation reduces gut inflammation, which may be both by stimulation of efferent (motor) pathways to the ENS, and stimulation of afferent pathways that connect to integrating centers in the CNS. Efferent pathways from the CNS have their anti-inflammatory effects through either or both vagal efferent neurons and sympathetic pathways. The final neurons in sympathetic pathways reduce gut inflammation by the action of noradrenaline on β2 adrenergic receptors expressed by immune cells. Activation of neural anti-inflammatory pathways is an attractive option to treat inflammatory bowel disease that is refractory to other treatments. Further investigation of the ways in which enteric reflexes, vagal pathways and sympathetic pathways integrate their effects to modulate the gut immune system and gut inflammation is needed to optimize neuromodulation therapy.

This protocol describes methods to dissociate nodose ganglion neurons from rats, identify and specifically collect neurons previously labeled using retrograde tracing, and then perform short read RNAseq analysis on pooled samples of small numbers of neurons or single neuronal cells. The parameters of the analysis are designed to allow deep sequencing (pooled cell samples, high numbers of reads per sample) to enable robust identification of genes expressed at low copy numbers that are important for neuronal function, such as ion channels and G-protein coupled receptors. It also includes a basic description of the sorts of differential expression analysis that can be performed on sequencing/transcriptomic data of this type.

The enteric nervous system is an important regulator of gastrointestinal, digestive and metabolic function. Here we describe protocols for clearing rat gastric tissue with the Clearing-enhanced 3D microscopy (Ce3D) method to increase optical clarity, to allow the use of thick tissue preparations for quantitation of the number and size of myenteric and submucosal ganglia in the rat fundus, corpus and antrum

The thickness, organization and relationships between the muscle layers of the rat stomach assist in understanding their function. Here we describe protocols for identifying, measuring and quantifying the muscle layers of the rat stomach using histological and microscopy techniques. We would like to acknowledge Phenomics Australia Histopathology and Slide Scanning Service, University of Melbourne.

The stomach acts as a buffer between the ingestion of food and its processing in the small intestine. It signals to the brain to modulate food intake and it in turn regulates the passage of a nutrient-rich fluid, containing partly digested food, into the duodenum. These processes need to be finely controlled, for example to restrict reflux into the esophagus and to transfer digesta to the duodenum at an appropriate rate. Thus, the efferent pathways that control gastric volume, gastric peristalsis and digestive juice production are critically important. We review these pathways with an emphasis on the identities of the final motor neurons and comparisons between species. The major types of motor neurons arising from gastric enteric ganglia are as follows: immunohistochemically distinguishable excitatory and inhibitory muscle motor neurons; four neuron types innervating mucosal effectors (parietal cells, chief cells, gastrin cells and somatostatin cells); and vasodilator neurons. Sympathetic efferent neurons innervate intramural arteries, myenteric ganglia and gastric muscle. Vagal efferent neurons with cell bodies in the brain stem do not directly innervate gastric effector tissues; they are pre-enteric neurons that innervate each type of gastric enteric motor neuron. The principal transmitters and co-transmitters of gastric motor neurons, as well as key immunohistochemical markers, are the same in rat, pig, human and other species.

Recordings of spike activity in small peripheral autonomic nerves close to organs can provide real‐time information about the functional states of those organs (afferent traffic) and the neural systems that control them (efferent traffic). However, maintaining long‐term multifiber recordings from autonomic nerves in vivo poses significant and unique technical challenges. Many visceral nerves consist almost entirely of C fiber axons in which action potentials produce very small currents with little spatial segregation, making individual units difficult to distinguish based on spike shape in multifiber filaments recorded conventionally under mineral oil, especially when signal‐to‐noise ratios are low. Ongoing activity can be due to hundreds of active axons, each firing at relatively low frequency. Slow changes in recording conditions can have large effects on the amplitudes of spikes and the signal‐to‐noise ratio over time, potentially causing spurious/artefactual changes in recorded spiking frequency when activity is quantified via thresholding. Previous approaches to drift compensation in recordings from neuronal somata or myelinated axons have utilized spatial information from multi‐electrode arrays or tracked changes in the amplitudes of single units differentiated by spike shape. Such approaches are not useful when recording the activity of C fibers due to their lack of spatial segregation and similar spike shapes. We have developed a method to track drift in signal and noise separately and to compensate for drift without the need for spike sorting. The method is robust to changes in the signal‐to‐noise ratio and can be applied online during data acquisition. It employs a recursive piecewise linear model that predicts resistance as a function of recording time. The implicit assumption is that the current source is constant and, conversely, the extracellular path resistance of the system is dynamic, being impacted by events such as mechanical disruption, or subtle movement of aqueous conducting fluid. We evaluated our method using recordings of gastrointestinal afferent axons during responses to inflammation. We also developed a simulation suite to generate artificial data that mimics the conditions in our recordings and a conventional spike sorting algorithm optimized to distinguish activity in single units and spike families in low noise C fiber recordings lasting hours. We have used these tools to validate the performance of our drift compensation algorithm and found it could compensate for drift effectively without spike sorting and could also improve the efficiency of spike sorting routines in long recordings where drift was present. The simulation suite, the analysis algorithms, and a graphical user interface are freely available via online repositories. We anticipate that, in future, as recording technologies improve, our algorithm will allow online assessment of activity in multi‐unit recordings in vivo that may provide a basis for closed‐loop therapy systems.Support or Funding InformationFunded by the Defense Advanced Research Projects Agency (DARPA) BTO, Contract No. N66001‐15‐2‐4060.

BACKGROUND:Peripheral autonomic nerves control visceral organs and convey information regarding their functional states and are, therefore, potential targets for new therapeutic and diagnostic approaches. Conventionally recorded multi-unit nerve activity in vivo undergoes slow differential drift of signal and noise amplitudes, making accurate monitoring of nerve activity for more than tens of minutes problematic.NEW METHOD:We describe an on-line drift compensation algorithm that utilizes recursive least-squares to estimate the relative change in spike amplitude due to changes in the nerve-electrode interface over time.RESULTS:We tested and refined our approach using simulated data and in vivo recordings from nerves supplying the small intestine under control conditions and in response to gut inflammation over several hours. The algorithm is robust to changes in recording conditions and signal-to-noise ratio and applicable to both single and multi-unit recordings. In uncompensated records, drift prevented "spike families" and single units from being discriminated accurately over hours. After rescaling, these were successfully tracked throughout recordings (up to 3 h).COMPARISON WITH EXISTING METHODS:Existing methods are subjective or compensate for drift using spatial information and spike shape data which is not practical in multi-unit peripheral nerve recordings. In contrast, this method is objective and applicable to data from a single differential multi-unit recording. In comparisons using simulated data the algorithm performed as well as or better than existing methods.CONCLUSIONS:Results suggest our drift compensation algorithm is widely applicable and robust, though conservative, when differentiating prolonged responses from drift in signal. Extracellular nerve recordings; drift compensation; chronic nerve recordings; closed-loop; multi-unit activity; spike discrimination; recursive least squares; real-time.

Disorders of gastric motility and sensation, including gastroparesis, are common, debilitating and currently poorly treated. To develop new treatments for these disorders, a thorough understanding of the gastric innervation and its pathology in humans is required. We have developed methods to identify and quantify the density of different classes of nerve fibers to the gastric muscle and mucosa. To do this we employed immunohistochemistry using characterized antibodies and a grid‐based quantitation method combined with high resolution confocal microscopy in resection tissue and biopsies from the gastric fundus and corpus. In the muscle we have identified nerve fibers immunoreactive for a range of neurochemical markers including neuronal nitric oxide synthase (NOS), vasoactive inhibitory peptide (VIP), tachykinins (TK), gastrin releasing peptide (GRP), neuropeptide Y (NPY), vesicular acetylcholine transporter (VAChT) and tyrosine hydroxylase (TH). Neuronal NOS and VIP were commonly colocalized in putative inhibitory nerves innervating the external musculature. We saw extensive labelling for VAChT and TK, both markers of the excitatory fibers innervating the muscle. GRP and NPY were also present in subgroups of excitatory nerve fibers. The density of inhibitory fibers (NOS) was greater than excitatory fibers (TK) in all external muscle layers. TH was found in a proportion of fibers in the muscle, suggesting direct sympathetic innervation of the muscle occurs in human stomach. These TH fibers also contained NPY but were separate from NPY immunoreactive excitatory muscle motor nerves.Prominent in mucosal innervation were fibers immunoreactive for VIP and GRP and a separate population containing VAChT. Another population of fibers containing NPY and TH were found around mucosal glands and also around arteries supplying the mucosa. For all common fiber types in the mucosa there was a gradient of densities such that most fibers were found in the bottom third of the mucosa and few fibers were found in the top third of the glands near the lumen. Rare CGRP containing fibers were also seen in the mucosa but were not present in every section suggesting they are rare and unevenly distributed.We conclude that the signature chemistries of nerve fibers that innervate the gastric muscle and mucosa in human are similar to those present in other species, including rat. An exception is the prominent direct innervation of the gastric muscle by extrinsic sympathetic axons which is often absent in small mammals. This parallel chemistry will allow us to extrapolate circuits determined experimentally in rat to human and to identify cell body types in the myenteric ganglia.Support or Funding InformationThis work was supported by NIH (SPARC) grant ID # OT2OD023847 (PI Terry Powley) to John Furness and Martin Stebbing.