Striving for humane deaths for laboratory mice: hypobaric hypoxia provides a potential alternative to carbon dioxide exposure.

Killing is often an unavoidable and necessary procedure for laboratory mice involved in scientific research, and providing a humane death is vital for public acceptance. Exposure to carbon dioxide (CO2) gas is the most widely used methodology despite well proven welfare concerns. Consequently, the continued use of CO2 and its globally permitted status in legislation and guidelines presents an ethical dilemma for users. We investigated whether killing with hypobaric hypoxia via gradual decompression was associated with better welfare outcomes for killing laboratory mice. We compared the spontaneous behaviour of mice exposed to CO2, decompression or sham conditions, and used analgesic or anxiolytic interventions to determine their relative welfare impact. Gradual decompression resulted in longer times to unconsciousness and death and the pharmacological interventions support the notion of a minimally negative animal experience, while providing further evidence for pain and anxiety associated with exposure to CO2. Decompression resulted in moderate ear haemorrhage, but our welfare assessment suggests this may happen when mice are unconscious. Hence, gradual decompression could be the basis of significant refinement for killing laboratory mice. Future work should corroborate behaviour with neurobiological markers of loss of consciousness to verify the conscious phase of concern for animal welfare.

Radon transport events associated with the impact of a NORM repository in the SW of Europe.

Two radon measurement stations located to the north and south of a NORM (Naturally Occurring Radioactive Materials) repository of phosphogypsum (southwest of Europe) were used to monitor radon behavior during 2018. The stations are located at opposing sides of the repository, one in Huelva City to the north and other one in a rural area to the south. This setup aimed to identify the influence of the NORM repository on each station and use radon levels as a marker of atmospheric transport in the local area. To achieve this, a comparison was carried out with other coastal stations in the south of Spain, finding higher average concentrations in Huelva City, ~3.3 Bq m-3. Hierarchical clustering was applied to identify days with different radon patterns at each Huelva station, detecting possible local radon transport events from the repository. Three events were investigated with WRF (Weather Research and Forecasting) and FLEXPART-WRF (FLEXible PARTicle dispersion model). It was found that both sampling sites required atmospheric stagnant conditions to reach high radon concentration. However, under these conditions the urban station showed high radon regardless of wind direction while the rural station also required radon transport from the repository, either directly or indirectly.

Resumen ejecutivo: Documento de posicionamiento: evaluación y manejo de la hipoglucemia en el paciente con diabetes mellitus 2020. Grupo de Trabajo de Diabetes Mellitus de la Sociedad Española de Endocrinología y Nutrición / Executive summary: Position document: Evaluation and management of hypoglycemia in the patient with diabetes mellitus 2020. Diabetes Mellitus Working Group of the Spanish Society of Endocrinology and Nutrition

Proporcionar unas recomendaciones prácticas para la evaluación y el manejo de la hipoglucemia en pacientes con diabetes mellitus. Miembros del Grupo de Trabajo de Diabetes Mellitus de la Sociedad Española de Endocrinología y Nutrición (SEEN). Las recomendaciones se formularon según el sistema Grading of Recommendations, Assessment, Development, and Evaluation (GRADE) para establecer tanto la fuerza de las recomendaciones como el grado de evidencia. Se realizó una búsqueda sistemática en MEDLINE (PubMed) de la evidencia disponible para cada tema, y se revisaron artículos escritos en inglés y castellano con fecha de inclusión hasta el 28 de febrero de 2020. En este resumen ejecutivo incluimos la evidencia reciente incorporada desde 2013. El documento establece unas recomendaciones prácticas basadas en la evidencia acerca de la evaluación y manejo de la hipoglucemia en pacientes con diabetes mellitus.

To provide practical recommendations for the evaluation and management of hypoglycemia in patients with diabetes mellitus. Members of the Diabetes Mellitus Working Group of the Spanish Society of Endocrinology and Nutrition (SEEN). The recommendations were made based on the Grading of Recommendations, Assessment, Development and Evaluation (GRADE) system to establish both the strength of the recommendations and the level of evidence. A systematic search was made in MEDLINE (PubMed) for the available evidence on each subject, and articles written in English and Spanish with an inclusion date up to 28 February 2020 were reviewed. This executive summary takes account of the evidence incorporated since 2013. The document establishes practical evidence-based recommendations regarding the evaluation and management of hypoglycemia in patients with diabetes mellitus.

Influence of the accumulation chamber insertion depth to measure surface radon exhalation rates.

A common method to measure radon exhalation rates relies on the accumulation chamber technique. Usually, this approach only considers one-dimensional gas transport within the soil that neglects lateral diffusion. However, this lateral transport could reduce the reliability of the method. In this work, several cylindrical-shaped accumulation chambers were built with different heights to test if the insertion depth of the chamber into the soil improves the reliability of the method and, in that case, if it could limit the radon lateral diffusion effects. To check this hypothesis in laboratory, two reference exhalation boxes were manufactured using phosphogypsum from a repository located nearby the city of Huelva, in the southwest of Spain. Laboratory experiments showed that insertion depth had a deep impact in reducing the effective decay constant of the system, extending the interval where the linear fitting can be applied, and consistently obtaining reliable exhalation measurements once a minimum insertion depth is employed. Field experiments carried out in the phosphogypsum repository showed that increasing the insertion depth could reduce the influence of external effects, increasing the repeatability of the method. These experiments provided a method to obtain consistent radon exhalation measurements over the phosphogypsum repository.

Occurrence, prevalence, and explanatory environmental variables of Spirocerca vulpis infestation in the foxes of western Spain.

The main aim of this study was to not only establish the prevalence of the recently described Spirocerca vulpis parasite in the wild-life cycle of carnivores in western Spain but to also elaborate a model to explain the risk of infestation based on 16 topo-climatic and habitat variables. During the period from June 2016 to November 2017, 1644 carcasses of red foxes (Vulpes vulpes) and another 105 wild mammals, legally hunted or killed in car accidents, were analyzed. Parasitic nodules of Spirocerca were found in 6% of the foxes, and the molecular analyses established a homology of our samples with the species S. vulpis. There were no differences in the occurrence of the infestation between sexes, but there were differences in terms of age, such that infestation was proportionally more frequent among young individuals. In terms of temporality, a higher percentage of positive cases was observed during the late-autumn and winter months, especially between December and February. This study provides new data on the factors that predispose S. vulpis infection in the red fox. Model results indicate that a spatial pattern exists in the occurrence and prevalence of this species in the studied area (higher probabilities to the west), and that this pattern seems to mainly be associated with topo-climatic variables.

Pathological consequences of low atmospheric pressure stunning in broiler chickens.

Low atmospheric pressure stunning (LAPS) is a novel approach to pre-slaughter stunning of chickens using progressive hypobaric hypoxia by the application of gradual decompression (280s cycle) according to a set of prescribed pressure curves. Low atmospheric pressure stunning produces a non-recovery state. Concerns have been raised relating to the possible pathological and welfare consequences of expansion of air in the body during LAPS. In a randomised trial, we compared the gross pathology of broilers exposed to LAPS with a control group euthanised by intravenous injection of pentobarbital sodium (60 mixed sex broilers per treatment). The birds were exposed to each treatment in triplets and all birds were subject to necropsy examination to detect and score (1 to 5, minimal to severe) haemorrhagic lesions or congestion for all major organs and cavities (e.g. air sacs, joints, ears and heart) as well as external assessment for product quality (e.g. wing tips). Behavioural data (latency to loss of posture and motionless) and chamber cycle data (temperature, humidity, pressure and oxygen availability) confirmed that LAPS had been applied in a manner representative of the commercial process. All of the organs observed were structurally intact for both treatment groups. No lesions were observed in the external ears, oral cavity, tracheal lumen, crop and air sacs of birds from either treatment group. There was no difference between treatments in the wingtips, nasal turbinates, thymus, biceps femoralis and colon. Haemorrhagic lesions were observed in the calvaria, brains, hearts and lungs of both treatment groups, but lesions in these areas were more severe in the LAPS treatment group. It was not possible to distinguish between pathological changes induced by decompression or recompression. In the barbiturate group, more severe haemorrhagic lesions were observed in the superficial pectoral muscles as well as greater congestion of the infraorbital sinuses, liver, spleens, duodenum, kidneys and gonads. These findings provide evidence that LAPS did not result in distension of the intestines and air sacs sufficient to cause changes, which were grossly visible on postmortem examination. There was also no evidence of barotrauma in the ears and sinuses. The pathological changes observed in the barbiturate treatment were as expected based on barbiturate toxicity. Low atmospheric pressure stunning appears to produce pathological changes by a variety of well-established mechanisms, and while these pathological data have limited value as welfare indicators, the results confirm that organ integrity was not compromised by the process.

Calcium isotopes offer clues on resource partitioning among Cretaceous predatory dinosaurs.

Large predators are overabundant in mid-Cretaceous continental dinosaur assemblages of North Africa. Such unbalanced ecosystem structure involves, among predatory dinosaurs, typical abelisaurid or carcharodontosaurid theropods co-occurring with long-snouted spinosaurids of debated ecology. Here, we report calcium (Ca) isotope values from tooth enamel (expressed as δ44/42Ca) to investigate resource partitioning in mid-Cretaceous assemblages from Niger (Gadoufaoua) and Morocco (Kem Kem Beds). In both assemblages, spinosaurids display a distinct isotopic signature, the most negative in our dataset. This distinct taxonomic clustering in Ca isotope values observed between spinosaurids and other predators provides unambiguous evidence for niche partitioning at the top of the trophic chains: spinosaurids foraged on aquatic environments while abelisaurid and carcharodontosaurid theropods relied almost exclusively on terrestrial resources.

Rare but evolutionarily consequential outcrossing in a highly inbred zoonotic parasite.

Recurrent self-mating can result in nearly clonal propagation of biological lineages, but even occasional outcrossing can serve to redistribute variation in future generations, providing cohesion among regional populations. The zoonotic parasite Trichinella spiralis has been suspected to undergo frequent inbreeding, resulting in genetically uniform larval cohorts which differ markedly from one another. Here, we explored the extent of inbreeding for this parasite by determining how genetic variation (at variable microsatellite markers) is distributed among 1379 larvae derived from 41 wild boars in Extremadura, Spain. In particular, we sought to determine how much of the genetic variation in this region's parasites occurs among the larvae of any given wild boar, and whether each derives from one, or more, parental lineages. We found strong evidence for inbreeding, resulting in genetically distinct parasite subpopulations among the parasites derived from many pairs of wild boar. Fully two-thirds of these parasite cohorts appear to derive from inbred parents; in 10% of the wild boars, parasites were so inbred as to become absolutely fixed in all of the assayed genetic loci. In spite of this, more than one pair of parents appear to have given rise to the infections in one-third of the sampled wild boars, resulting in mixed infections. These mixed infections should slow losses of heterozygosity and multi-locus polymorphism in any given parasite lineage. Such outcrossing should limit distinctions that would otherwise accumulate among transmission chains, thereby enforcing cohesion through the region's population in spite of its marked departure from panmixia. Conditions of transmission may differ in other regions, where such epidemiological features may engender different evolutionary outcomes.

Developing a new Bayesian Risk Index for risk evaluation of soil contamination.

Industrial and agricultural activities heavily constrain soil quality. Potentially Toxic Elements (PTEs) are a threat to public health and the environment alike. In this regard, the identification of areas that require remediation is crucial. In the herein research a geochemical dataset (230 samples) comprising 14 elements (Cu, Pb, Zn, Ag, Ni, Mn, Fe, As, Cd, V, Cr, Ti, Al and S) was gathered throughout eight different zones distinguished by their main activity, namely, recreational, agriculture/livestock and heavy industry in the Avilés Estuary (North of Spain). Then a stratified systematic sampling method was used at short, medium, and long distances from each zone to obtain a representative picture of the total variability of the selected attributes. The information was then combined in four risk classes (Low, Moderate, High, Remediation) following reference values from several sediment quality guidelines (SQGs). A Bayesian analysis, inferred for each zone, allowed the characterization of PTEs correlations, the unsupervised learning network technique proving to be the best fit. Based on the Bayesian network structure obtained, Pb, As and Mn were selected as key contamination parameters. For these 3 elements, the conditional probability obtained was allocated to each observed point, and a simple, direct index (Bayesian Risk Index-BRI) was constructed as a linear rating of the pre-defined risk classes weighted by the previously obtained probability. Finally, the BRI underwent geostatistical modeling. One hundred Sequential Gaussian Simulations (SGS) were computed. The Mean Image and the Standard Deviation maps were obtained, allowing the definition of High/Low risk clusters (Local G clustering) and the computation of spatial uncertainty. High-risk clusters are mainly distributed within the area with the highest altitude (agriculture/livestock) showing an associated low spatial uncertainty, clearly indicating the need for remediation. Atmospheric emissions, mainly derived from the metallurgical industry, contribute to soil contamination by PTEs.

Evaluation of the potential killing performance of novel percussive and cervical dislocation tools in chicken cadavers.

1. Four mechanical poultry killing devices; modified Armadillo (MARM), modified Rabbit Zinger (MZIN), modified pliers (MPLI) and a novel mechanical cervical dislocation (NMCD) gloved device, were assessed for their killing potential in the cadavers of euthanised birds. 2. A 4 × 4 × 4 factorial design (batch × device × bird type + age) was employed. Ten bird cadavers per bird type and age were tested with each of the 4 devices (N = 160 birds). All cadavers were examined post-mortem to establish the anatomical damage caused. 3. NMCD, MARM and MZIN demonstrated killing potential, as well as consistency in their anatomical effects. NMCD had the highest killing potential, with 100% of birds sustaining the required physical trauma to have caused rapid death. 4. The MPLI was inconsistent, and only performed optimally for 27.5% of birds. Severe crushing injury was seen in >50% of MPLI birds, suggesting that birds would die of asphyxia rather than cerebral ischaemia, a major welfare concern. As a result, the MPLI are not recommended as a humane on-farm killing device for chickens. 5. This experiment provides important data on the killing potential of untried novel percussive and mechanical cervical dislocation methods, informing future studies.

Detection of Leishmania DNA and blood meal sources in phlebotomine sand flies (Diptera: Psychodidae) in western of Spain: Update on distribution and risk factors associated.

Leishmaniosis caused by Leishmania infantum is present in Mediterranean countries, with high prevalence in areas of the center and south of Spain. However, in some regions such as Extremadura (in southwest of Spain), data has not been updated since 1997. The aim of this work was (i) to provide information about the distribution of phlebotomine sand fly species in western of Spain (Extremadura region), (ii) to determine risk factors for the presence of sand fly vectors and (iii) to detect Leishmania DNA and identify blood meal sources in wild caught females. During 2012-2013, sand flies were surveyed using CDC miniature light-traps in 13 of 20 counties in Extremadura. Specimens were identified morphologically and females were used for molecular detection of Leishmania DNA by kDNA, ITS-1 and cyt-B. In addition, blood meals origins were analyzed by a PCR based in vertebrate cyt b gene. A total of 1083 sand flies of both gender were captured and identified. Five species were collected, Phlebotomus perniciosus (60.76%), Sergentomyia minuta (29.92%), P. ariasi (7.11%), P. papatasi (1.48%) and P. sergenti (0.74%). The last three species constitute the first report in Badajoz, the most southern province of Extremadura region. Leishmania DNA was detected in three out of 435 females (one P. pernicious and two S. minuta). Characterization of obtained DNA sequences by phylogenetic analyses revealed close relatedness with Leishmania tarentolae in S. minuta and L. infantum in P. perniciosus. Haematic preferences showed a wide range of hosts, namely: swine, humans, sheep, rabbits, horses, donkeys and turkeys. The simultaneous presence of P. perniciosus and P. ariasi vectors, the analysis of blood meals, together with the detection of L. infantum and in S. minuta of L. tarentolae, confirms the ideal conditions for the transmission of this parasitosis in the western of Spain. These results improve the epidemiological knowledge of leishmaniosis and its vectors in this part of Spain, highlighting the need for ongoing entomological and parasitological surveillance.

Effects of light on responses to low atmospheric pressure stunning in broilers.

Low atmospheric pressure stunning (LAPS) is a novel approach to poultry stunning involving the application of gradual decompression lasting 280 s according to a prescribed pressure curve. The aim of this study was to determine how behavioural, electroencephalogram (EEG) and electrocardiogram (ECG) responses to LAPS are influenced by illumination of the decompression chamber. A secondary aim was to examine responses to the decompression chamber without LAPS being applied, as such a "sham" control has been absent in previous studies. A two by two factorial design was employed, with LAPS/light, LAPS/dark, sham/light and sham/dark treatments (N = 20 per treatment). Broilers were exposed to each treatment in pairs, in each of which one bird was instrumented for recording EEG and ECG. Illumination was applied at 500 lx, and in sham treatments, birds were identically handled but remained undisturbed in the LAPS chamber without decompression for 280 s. Birds which underwent the sham treatment exhibited behaviours which were also observed in LAPS (e.g. sitting) while those exposed to LAPS exhibited hypoxia-related behaviours (e.g. ataxia, loss of posture). Behavioural latencies and durations were increased in the sham treatments, since the whole cycle time was available (in LAPS; birds were motionless by 186 s). Within the sham treatments, illumination increased active behaviour and darkness induced sleep, but slow-wave EEG was seen in both. The pattern of EEG response to LAPS (steep reduction in median frequency in the first 60 s and increased total power) was similar, irrespective of illumination, though birds in darkness had shorter latencies to loss of consciousness and isoelectric EEG. Cardiac responses to LAPS (pronounced bradycardia) closely matched those reported previously and were not affected by illumination. The effects of LAPS/sham treatment primarily reflected the presence/absence of hypoxia, while illumination affected activity/sleep levels in sham-treated birds and slowed time to unconsciousness in birds undergoing LAPS. Therefore, it is recommended that LAPS be conducted in darkness for poultry.

Detection of Zoonotic Protozoa Toxoplasma gondii and Sarcocystis suihominis in Wild Boars from Spain.

Food safety regulations require the control of the presence of protozoa in meats destined for human consumption. Wild boar (Sus scrofa) meat may constitute a source of zoonoses. A 23.8% (688/2881) seroprevalence of anti-Toxoplasma gondii antibodies and 72.2% (662/910) Sarcocystis sarcocysts prevalence were detected among wild boars hunted in Southwestern areas of Spain. Identity of Sarcocystis spp. was performed by RFLP-PCR and sequencing, detecting S. miescheriana (7/8) and the zoonotic S. suihominis (1/8). Risk assessment studies of these coccidian in meats destined to human consumption are needed.

Trace metal partitioning over a tidal cycle in an estuary affected by acid mine drainage (Tinto estuary, SW Spain).

The Tinto River estuary is highly polluted with the acid lixiviates from old sulphide mines. In this work the behaviour of dissolved and particulate trace metals under strong chemical gradients during a tidal cycle is studied. The pH values range from 4.4 with low tide to 6.9 with high tide. Precipitation of Fe and Al is intense during rising tides and As and Pb are almost exclusively found in the particulate matter (PM). Sorption processes are very important in controlling the mobility (and hence bioavailability) of some metals and particularly affect Cu below pH 6. Above pH~6 Cu is desorbed, probably by the formation of Cu(I)-chloride complexes. Although less pronounced than Cu, also Zn desorption above pH 6.5 seems to occur. Mn and Co are affected by sorption processes at pH higher than ca. 6. Cd behaves conservatively and Ni is slightly affected by sorption processes.

Chronic constipation in the elderly: an unusual presentation of colonic dysmotility in an elderly patient.

Introduction. Chronic constipation is common in the elderly, and often no underlying pathology is found. Primary colonic dysmotility has been described in children but is rare in the elderly. Case report. We present an 82-year-old female with long standing constipation presenting acutely with large bowel obstruction. Laparotomy and Hartman's procedure was performed, and a grossly distended sigmoid colon was resected. Histology revealed a primary myopathic process. Conclusion. Primary colonic myopathy should be considered in elderly patients presenting with large bowel obstruction and a long preceding history of constipation, particularly when previous endoscopic examinations were normal.

Uranium behaviour in an estuary polluted by mining and industrial effluents: the Ría of Huelva (SW of Spain).

This paper describes a comprehensive study of the behaviour of U in the Ría of Huelva estuary, formed by the Tinto and Odiel rivers. This ecosystem is conditioned by two hydrochemical facts: one connected with the acid mining drainage (AMD) generated in the first section of the river basins, and another one related to the fertilizer industry located at the estuary. AMD gives a singular character to these rivers; low pH and high redox potential that keep high amounts of toxic elements and radionuclides in dissolution. Most of the data for dissolved U in estuaries indicate conservative mixing, but there are examples of non-conservative behaviour attributed to oxidation/reduction processes or solubility variations. In the Ría of Huelva estuary the U shows a non-conservative behaviour due to solubility changes produced by variations in the pH. A complete removal of riverine dissolved U is observed in a pH range of 4-6. At higher pH values, U release from suspended matter, and probably also from sediments into the dissolved phase is found.

Fetal haemopoiesis marking low-grade urinary bladder cancer.

BACKGROUND: The immunohistochemical features of fetal haemoglobin cells and their distribution patterns in solid tumours, such as colorectal cancer and blastomas, suggest that fetal haemopoiesis may take place in these tumour tissues. These locally highly concentrated fetal haemoglobin (HbF) cells may promote tumour growth by providing a more efficient oxygen supply. METHODS AND RESULTS: Biomarkers of HbF were checked in transitional cell carcinoma (TCC) of the urinary bladder, assessing this as a new parameter for disease management. Fetal haemoglobin was immunohistochemically examined in tumours from 60 patients with TCC of the bladder. Fetal haemoglobin erythrocytes and erythroblasts were mainly clonally distributed in proliferating blood vessels and not mixed with normal haemoglobin erythrocytes. The proportion of such HbF blood vessels could reach more than half of the total number of vessels. There were often many HbF erythroblasts distributed in one-cell or two-cell capillaries and present as 5-15% of cells in multi-cell vessels. This suggests a local proliferation of HbF-cell progenitors. Fetal haemoglobin cells were prominently marking lower grades of tumours, as 76% (n=21) of the patients with G1pTa were HbF+, whereas only 6.7% (n=30) of the patients with G3pT1-pT2a were HbF+. CONCLUSION: Our results suggest that HbF, besides being a potential new marker for early tumour detection, might be an essential factor of early tumour development, as in fetal life. Inhibiting HbF upregulation may provide a therapeutic target for the inhibition of tumour growth.

Voltage-gated potassium channel (K(v) 1) autoantibodies in patients with chagasic gut dysmotility and distribution of K(v) 1 channels in human enteric neuromusculature (autoantibodies in GI dysmotility).

BACKGROUND: Autoantibodies directed against specific neuronal antigens are found in a significant number of patients with gastrointestinal neuromuscular diseases (GINMDs) secondary to neoplasia. This study examined the presence of antineuronal antibodies in idiopathic GINMD and GINMD secondary to South American Trypanosomiasis. The GI distribution of voltage-gated potassium channels (VGKCs) was also investigated. METHODS: Seventy-three patients were included in the study with diagnoses of primary achalasia, enteric dysmotility, chronic intestinal pseudo-obstruction, esophageal or colonic dysmotility secondary to Chagas' disease. Sera were screened for specific antibodies to glutamic acid decarboxylase, voltage-gated calcium channels (VGCCs; P/Q subtype), nicotinic acetylcholine receptors (nAChRs; α3 subtype), and voltage-gated potassium channels (VGKCs, K(V) 1 subtype) using validated immunoprecipitation assays. The distribution of six VGKC subunits (K(V) 1.1-1.6), including those known to be antigenic targets of anti-VGKC antibodies was immunohistochemically investigated in all main human GI tract regions. KEY RESULTS: Three patients (14%) with chagasic GI dysmotility were found to have positive anti-VGKC antibody titers. No antibodies were detected in patients with idiopathic GINMD. The VGKCs were found in enteric neurons at every level of the gut in unique yet overlapping distributions. The VGKC expression in GI smooth muscle was found to be limited to the esophagus. CONCLUSIONS & INFERENCES: A small proportion of patients with GI dysfunction secondary to Chagas' disease have antibodies against VGKCs. The presence of these channels in the human enteric nervous system may have pathological relevance to the growing number of GINMDs with which anti-VGKC antibodies have been associated.

Regional- and agonist-dependent facilitation of human neurogastrointestinal functions by motilin receptor agonists.

BACKGROUND AND PURPOSE: Delayed gastric emptying is poorly managed. Motilin agonists are potential treatments but inadequate understanding into how enteric nerve functions are stimulated compromises drug/dose selection. Resolution is hampered by extreme species dependency so methods were developed to study human gastrointestinal neuromuscular activities and the neurobiology of motilin. EXPERIMENTAL APPROACH: Protocols to study neuromuscular activities were developed for different regions of human stomach and intestine (71 patients) using circular muscle preparations and electrical field stimulation (EFS) of intrinsic nerves. Other tissues were fixed for immunohistochemistry. KEY RESULTS: EFS evoked contractions and/or relaxations via cholinergic and nitrergic neurons, with additional tachykinergic activity in colon; these were consistent after 154 min (longer if stored overnight). Motilin 1-300 nM and the selective motilin agonist GSK962040 0.1-30 µM acted pre-junctionally to strongly facilitate cholinergic contractions of the antrum (E(max) ≈ 1000% for motilin), with smaller increases in fundus, duodenum and ileum; high concentrations increased baseline muscle tension in fundus and small intestine. There were minimal effects in the colon. In the antrum, cholinergic facilitation by motilin faded irregularly, even with peptidase inhibitors, whereas facilitation by GSK962040 was long lasting. Motilin receptor immunoreactivity was identified in muscle and myenteric plexus predominantly in the upper gut, co-expressed with choline acetyltransferase in neurons. CONCLUSIONS AND IMPLICATIONS: Motilin and GSK962040 strongly facilitated cholinergic activity in the antrum, with lower activity in fundus and small intestine only. Facilitation by motilin was short lived, consistent with participation in migrating motor complexes. Long-lasting facilitation by GSK962040 suggests different receptor interactions and potential for clinical evaluation.