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Background: Felcisetrag (5-hydroxytryptamine-4 receptor [5-HT 4 ] agonist) is under investigation as prophylaxis or active treatment for accelerating resolution of gastrointestinal function post-surgery., Methods: Phase 2, randomized, placebo-controlled, parallel five-arm, double-blind, multicenter study (NCT03827655) in 209 adults undergoing open or laparoscopic-assisted bowel surgery. Patients received intravenous placebo, felcisetrag 0.1 mg/100 ​mL or 0.5 mg/100 ​mL pre-surgery only, or pre-surgery and daily post-surgery until return of gastrointestinal function or for up to 10 days., Primary Endpoint: time to recovery of gastrointestinal function., Results: Median time to recovery of gastrointestinal function was 2.6 days for both felcisetrag 0.5 ​mg daily and 0.5 ​mg pre-surgery versus 1.9 days for placebo (p ​> ​0.05). There were no notable differences in adverse events between treatment arms., Conclusions: Felcisetrag was well tolerated with no new safety concerns. However, no clinically meaningful difference in time to recovery of gastrointestinal function versus placebo was observed. Further investigation of the utility of 5-HT 4 agonists in complicated, open abdominal surgeries may be warranted., Competing Interests: Declaration of competing interest GB has received a research grant from Takeda and is an Editorial Board Member for Gut. SA has nothing to declare. ME has nothing to declare. GD was an employee of Takeda Development Center Americas, Inc. and received stock or stock options at the time of the study; they are currently an employee of Ironwood Pharmaceuticals, Inc. RG, PK, JT, PT, JW, and YZ are employees of Takeda Development Center Americas, Inc. and receive stock or stock options. PRW was an employee of Takeda Development Center Americas, Inc. and received stock or stock options at the time of the study; he is currently the founding principal at Second Brain Consulting, LLC, Philadelphia, PA, USA., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

A key goal of conservation is to protect biodiversity by supporting the long-term persistence of viable, natural populations of wild species. Conservation practice has long been guided by genetic, ecological and demographic indicators of risk. Emerging evidence of animal culture across diverse taxa and its role as a driver of evolutionary diversification, population structure and demographic processes may be essential for augmenting these conventional conservation approaches and decision-making. Animal culture was the focus of a ground-breaking resolution under the Convention on the Conservation of Migratory Species of Wild Animals (CMS), an international treaty operating under the UN Environment Programme. Here, we synthesize existing evidence to demonstrate how social learning and animal culture interact with processes important to conservation management. Specifically, we explore how social learning might influence population viability and be an important resource in response to anthropogenic change, and provide examples of how it can result in phenotypically distinct units with different, socially learnt behavioural strategies. While identifying culture and social learning can be challenging, indirect identification and parsimonious inferences may be informative. Finally, we identify relevant methodologies and provide a framework for viewing behavioural data through a cultural lens which might provide new insights for conservation management.

DNA methylation data facilitate the development of accurate molecular estimators of chronological age or "epigenetic clocks." We present a robust epigenetic clock for the beluga whale, Delphinapterus leucas , developed for an endangered population in Cook Inlet, Alaska, USA. We used a custom methylation array to measure methylation levels at 37,491 cytosine-guanine sites (CpGs) from skin samples of dead whales ( n  = 67) whose chronological ages were estimated based on tooth growth layer groups. Using these calibration data, a penalized regression model selected 23 CpGs, providing an R 2  = 0.92 for the training data; and an R 2  = 0.74 and median absolute age error = 2.9 years for the leave one out cross-validation. We applied the epigenetic clock to an independent dataset of 38 skin samples collected with a biopsy dart from living whales between 2016 and 2018. Age estimates ranged from 11 to 27 years. We also report sex correlations in CpG data and describe an approach of identifying the sex of an animal using DNA methylation. The epigenetic estimators of age and sex presented here have broad applications for conservation and management of Cook Inlet beluga whales and potentially other cetaceans., Competing Interests: SH is a founder of the non‐profit Epigenetic Clock Development Foundation which plans to license several of his patents from his employer UC Regents. The other authors declare no conflicts of interest., (© 2021 The Authors. Evolutionary Applications published by John Wiley & Sons Ltd.)

Background: Host-specific microbiomes play an important role in individual health and ecology; in marine mammals, epidermal microbiomes may be a protective barrier between the host and its aqueous environment. Understanding these epidermal-associated microbial communities, and their ecological- or health-driven variability, is the first step toward developing health indices for rapid assessment of individual or population health. In Cook Inlet, Alaska, an endangered population of beluga whales (Delphinapterus leucas) numbers fewer than 300 animals and continues to decline, despite more than a decade of conservation effort. Characterizing the epidermal microbiome of this species could provide insight into the ecology and health of this endangered population and allow the development of minimally invasive health indicators based on tissue samples., Results: We sequenced the hypervariable IV region of bacterial and archaeal SSU rRNA genes from epidermal tissue samples collected from endangered Cook Inlet beluga whales (n = 33) and the nearest neighboring population in Bristol Bay (n = 39) between 2012 and 2018. We examined the sequences using amplicon sequence variant (ASV)-based analyses, and no ASVs were associated with all individuals, indicating a greater degree of epidermal microbiome variability among beluga whales than in previously studied cetacean species and suggesting the absence of a species-specific core microbiome. Epidermal microbiome composition differed significantly between populations and across sampling years. Comparing the microbiomes of Bristol Bay individuals of known health status revealed 11 ASVs associated with potential pathogens that differed in abundance between healthy individuals and those with skin lesions or dermatitis. Molting and non-molting individuals also differed significantly in microbial diversity and the abundance of potential pathogen-associated ASVs, indicating the importance of molting in maintaining skin health., Conclusions: We provide novel insights into the dynamics of Alaskan beluga whale epidermal microbial communities. A core epidermal microbiome was not identified across all animals. We characterize microbial dynamics related to population, sampling year and health state including level of skin molting. The results of this study provide a basis for future work to understand the role of the skin microbiome in beluga whale health and to develop health indices for management of the endangered Cook Inlet beluga whales, and cetaceans more broadly.

Organochlorine (OC) profiles have been used as chemical "fingerprints" to infer an animal's foraging area. North Pacific killer whale (Orcinus orca) populations are exposed to different levels and patterns of OCs based on their prey, distribution, and amount of time spent in a particular area. To characterize concentrations and profiles of OCs found in various populations of North Pacific killer whales, polychlorinated biphenyls (PCBs), including dioxin-like congeners, DDTs, and hexachlorobenzene (HCB), were measured in biopsy blubber samples of photo-identified resident (fish-eating) and transient (mammal-eating) killer whales collected from 1994 through 2002 from Russian Far East waters to the waters of the west coast of the United States, representing 10 populations. We compared blubber OC concentrations based on ecotype (resident vs. transient), sex and reproductive maturity, and geographic area. We also examined OC mixtures to determine if we could detect segregated geographical areas (foraging areas) among the six populations with sufficient sample sizes. Transients had significantly higher OC concentrations than residents and adult male whales had consistently higher OC levels compared to adult females, regardless of ecotype. Our OC profile findings indicate segregated foraging areas for the North Pacific killer whales, consistent with observations of their geographic distributions. Several potential health risks have also been associated with exposure to high levels of contaminants in top-level predators including reproductive impairment, immune suppression, skeletal deformities, and carcinoma. The results of this baseline study provide information on the geographic distribution of OCs found in North Pacific killer whales, results which are crucial for assessing the potential health risks associated with OC exposure in this species., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Published by Elsevier B.V.)

Patients with chronic constipation benefit from treatment with 5-hydroxytryptamine 4 (5-HT 4 ) receptor agonists. However, the first-generation 5-HT 4 receptor agonists cisapride and tegaserod were withdrawn from the market owing to rare cardiovascular adverse events that were not 5-HT 4 -receptor-related but due to the lack of selectivity of these drugs. Here we report the nonclinical cardiovascular profile of the selective 5-HT 4 receptor agonist prucalopride. To assess its non-5-HT 4 receptor-mediated effects on cardiovascular electrophysiological parameters, in vitro studies were performed in human ether-à-go-go-related gene-transfected cells, guinea pig ventricular myocytes and papillary muscle preparations, rabbit and dog Purkinje fibers, and the Langendorff rabbit heart. In vivo experiments were performed in a rabbit model for drug-induced proarrhythmogenesis, in anesthetized guinea pigs, and anesthetized and conscious dogs. In addition, human platelet aggregation and coronary artery contraction were studied to exclude interactions that have been suggested to mediate the cardiovascular effects of tegaserod. Effects at 5-HT 4 receptors were evaluated in piglet and human atrial myocardium, and in anesthetized pigs. Finally, cardiovascular endpoints were investigated in chronic, repeated-dose toxicology studies at very high prucalopride doses in rats and dogs. No relevant effects were observed in any of the cardiovascular studies at concentrations at least 50 times the therapeutic plasma level. Only in pigs were minor and transient increases in heart rate and blood pressure noted upon first exposure to prucalopride, at plasma levels at least 10 times higher than human therapeutic plasma levels. Prucalopride may thus provide therapeutic benefit without the cardiovascular risks reported for other 5-HT 4 receptor agonists., (Copyright © 2018 The Author(s).)

Global climate change during the Late Pleistocene periodically encroached and then released habitat during the glacial cycles, causing range expansions and contractions in some species. These dynamics have played a major role in geographic radiations, diversification and speciation. We investigate these dynamics in the most widely distributed of marine mammals, the killer whale (Orcinus orca), using a global data set of over 450 samples. This marine top predator inhabits coastal and pelagic ecosystems ranging from the ice edge to the tropics, often exhibiting ecological, behavioural and morphological variation suggestive of local adaptation accompanied by reproductive isolation. Results suggest a rapid global radiation occurred over the last 350 000 years. Based on habitat models, we estimated there was only a 15% global contraction of core suitable habitat during the last glacial maximum, and the resources appeared to sustain a constant global effective female population size throughout the Late Pleistocene. Reconstruction of the ancestral phylogeography highlighted the high mobility of this species, identifying 22 strongly supported long-range dispersal events including interoceanic and interhemispheric movement. Despite this propensity for geographic dispersal, the increased sampling of this study uncovered very few potential examples of ancestral dispersal among ecotypes. Concordance of nuclear and mitochondrial data further confirms genetic cohesiveness, with little or no current gene flow among sympatric ecotypes. Taken as a whole, our data suggest that the glacial cycles influenced local populations in different ways, with no clear global pattern, but with secondary contact among lineages following long-range dispersal as a potential mechanism driving ecological diversification., (© 2015 John Wiley & Sons Ltd.)

The difficulties associated with detecting population boundaries have long constrained the conservation and management of highly mobile, wide-ranging marine species, such as killer whales (Orcinus orca). In this study, we use data from 26 nuclear microsatellite loci and mitochondrial DNA sequences (988bp) to test a priori hypotheses about population subdivisions generated from a decade of killer whale surveys across the northern North Pacific. A total of 462 remote skin biopsies were collected from wild killer whales primarily between 2001 and 2010 from the northern Gulf of Alaska to the Sea of Okhotsk, representing both the piscivorous "resident" and the mammal-eating "transient" (or Bigg's) killer whales. Divergence of the 2 ecotypes was supported by both mtDNA and microsatellites. Geographic patterns of genetic differentiation were supported by significant regions of genetic discontinuity, providing evidence of population structuring within both ecotypes and corroborating direct observations of restricted movements of individual whales. In the Aleutian Islands (Alaska), subpopulations, or groups with significantly different mtDNA and microsatellite allele frequencies, were largely delimited by major oceanographic boundaries for resident killer whales. Although Amchitka Pass represented a major subdivision for transient killer whales between the central and western Aleutian Islands, several smaller subpopulations were evident throughout the eastern Aleutians and Bering Sea. Support for seasonally sympatric transient subpopulations around Unimak Island suggests isolating mechanisms other than geographic distance within this highly mobile top predator.

A small set of acyclic analogs 5 were prepared to explore their structure-activity relationships (SARs) relative to heterocyclic core, opioid receptor (OR) agonists 4. Compound 5l was found to have very favorable OR binding affinities at the δ and μ ORs (r K(i) δ=1.3 nM; r K(i) μ=0.9 nM; h K(i) μ=1.7 nM), with less affinity for the κ OR (gp K(i) κ=55 nM). The OR functional profile for 5l varied from the previously described dual δ/μ OR agonists 4, with 5l being a potent, mixed dual δ OR antagonist/μ OR agonist [δ IC(50)=89 nM (HVD); μ EC(50)=1 nM (GPI); κ EC(50)=1.6 μM (GPC)]. Compound 5l has progressed through a clinical Phase II Proof of Concept study on 800 patients suffering from diarrhea-predominant Irritable Bowel Syndrome (IBS-d). This Phase II study was recently completed successfully, with 5l demonstrating statistically significant efficacy over placebo., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

The North Pacific right whale (Eubalaena japonica) was heavily exploited by both nineteenth century whaling and recent (1960s) illegal Soviet catches. Today, the species remains extremely rare especially in the eastern North Pacific. Here, we use photographic and genotype data to calculate the first mark-recapture estimates of abundance for right whales in the Bering Sea and Aleutian Islands. The estimates were very similar: photographic = 31 (95% CL 23-54), genotyping = 28 (95% CL 24-42). We also estimated the population contains eight females (95% CL 7-18) and 20 males (95% CL 17-37). Although these estimates may relate to a Bering Sea subpopulation, other data suggest that the total eastern North Pacific population is unlikely to be much larger. Its precarious status today-the world's smallest whale population for which an abundance estimate exists-is a direct consequence of uncontrolled and illegal whaling, and highlights the past failure of international management to prevent such abuses.

The release of small intestinal hormones by constituents of ingested food, such as fatty acids, is integral to post-prandial responses that reduce food intake. Recent evidence suggests that small intestinal electrical stimulation reduces food intake, although the mechanism of action is debated. To test the hypothesis that intestinal stimulation directly alters hormone release locally we used isolated rat distal ileum and measured glucagon-like peptide-1 (GLP-1) released in the presence or absence of linoleic acid (LA) and electrical field stimulation (EFS). Intact segments were oriented longitudinally between bipolar stimulating electrodes in organ bath chambers containing modified Krebs-Ringers bicarbonate (KRB) buffer including protease inhibitors. Incubation in LA (3 mg ml(-1)) for 45 min increased GLP-1 concentration (21.9 +/- 2.6 pM versus KRB buffer alone 3.6 +/- 0.1 pM). Eleven electrical stimulation conditions were tested. In the presence of LA none of the stimulation conditions inhibited LA-evoked GLP-1 release, whereas two high frequency short pulse widths (14 V, 20 Hz, 5 ms and 14 V, 40 Hz, 5 ms) and one low frequency long pulse width (14 V, 0.4 Hz, 300 ms) EFS conditions enhanced LA-evoked GLP-1 release by >250%. These results are consistent with a local effect of intestinal electrical stimulation to enhance GLP-1 release in response to luminal nutrients in the intestines. Enhancing hormone release could improve the efficacy of intestinal electrical stimulation and provide a potential treatment for obesity and metabolic conditions.

Background: Prokineticins 1 and 2 (PROK1 and PROK2) are so named because they contract gastrointestinal smooth muscle, yet little else is known about their role in gastrointestinal function. Therefore, we used a combination of approaches to elucidate the mechanisms by which PROK1 alters ileal contractility and secretion in rats., Methods: RT-PCR and immunofluorescence were used to determine PROK and receptor (PK-R) mRNA levels and PK-R1 localization, respectively. Upper GI transit and fluid secretion were determined in vivo. Contractility and intestinal epithelial ion transport were assessed in isolated ileal segments., Key Results: In the gastric fundus, PROK1 mRNA is highly expressed (70-fold >PROK2 mRNA) whereas the ileum has the highest mRNA expression of its receptor. PK-R1 immunoreactivity is visualized in ileal crypt cells, and in submucosal and myenteric neurons. In ileal segments, PROK1 evokes biphasic contractile responses consisting of an early, TTX-sensitive response (EC(50) = 87.8 nmol L(-1)) followed by a late, TTX-insensitive (EC(50) = 72.4 nmol L(-1)) component that is abolished in mucosa-free preparations. Oral administration of PROK1 enhances small bowel transit (111 +/- 3% of control) and fluid secretion (340 +/- 90% of control) and in muscle-stripped ileal preparations increases short-circuit current (EC(50) = 8.2 nmol L(-1)) in a TTX-insensitive manner. The PROK1-evoked Cl- secretion is reduced by piroxicam (non-selective cyclooxygenase inhibitor), and a prostaglandin EP(4) receptor antagonist (AH23848), but not a thromboxane receptor antagonist (GR32191B)., Conclusions & Inferences: These results demonstrate that PROK1 has oral prokinetic and secretogogue activity and that it acts on the intestinal mucosa via PK-R1 and prostaglandin receptors to mediate these effects.

Seasonal feeding behavior and high fidelity to feeding areas allow humpback whales (Megaptera novaeangliae) to be used as biological indicators of regional contamination. Biopsy blubber samples from male individuals (n = 67) were collected through SPLASH, a multinational research project, in eight North Pacific feeding grounds. Additional male samples (n = 20) were collected from one North Atlantic feeding ground. Persistent organic pollutants were measured in the samples and used to assess contaminant distribution in the study areas. North Atlantic (Gulf of Maine) whales were more contaminated than North Pacific whales, showing the highest levels of polychlorinated biphenyls (PCBs), polybrominated diphenyl ethers (PBDEs), and chlordanes. The highest dichlorodiphenyltrichloroethane (DDT) levels were detected in whales feeding off southern California, USA. High-latitude regions were characterized by elevated levels of hexachlorocyclohexanes (HCHs) but generally nondetectable concentrations of PBDEs. Age was shown to have a positive relationship with SigmaPCBs, SigmaDDTs, Sigmachlordanes, and total percent lipid. Contaminant levels in humpback whales were comparable to other mysticetes and lower than those found in odontocete cetaceans and pinnipeds. Although these concentrations likely do not represent a significant conservation threat, levels in the Gulf of Maine and southern California may warrant further study., ((c) 2009 SETAC.)

Mu-opioid analgesics are a mainstay in the treatment of acute and chronic pain of multiple origins, but their side effects, such as constipation, respiratory depression, and abuse liability, adversely affect patients. The recent demonstration of the up-regulation and membrane targeting of the delta-opioid receptor (DOR) following inflammation and the consequent enhanced therapeutic effect of delta-opioid agonists have enlivened the search for delta-opioid analgesic agents. JNJ-20788560 [9-(8-azabicyclo-[3.2.1]oct-3-ylidene)-9H-xanthene-3-carboxylic acid diethylamide] had an affinity of 2.0 nM for DOR (rat brain cortex binding assay) and a naltrindole sensitive DOR potency of 5.6 nM (5'-O-(3-[(35)S]thio)triphosphate assay). The compound had a potency of 7.6 mg/kg p.o. in a rat zymosan radiant heat test and of 13.5 mg/kg p.o. in a rat Complete Freund's adjuvant RH test but was virtually inactive in an uninflamed radiant heat test. In limited studies, tolerance was not observed to the antihyperalgesic or antinociceptive effects of the compound. Unlike ibuprofen, JNJ-20788560 did not produce gastrointestinal (GI) erosion. Although morphine reduced GI motility at all doses tested and reached nearly full effect at the highest dose, JNJ-20788560 did not retard transit at the lowest dose and reached only 11% reduction at the highest dose administered. Unlike morphine, JNJ-20788560 did not exhibit respiratory depression (blood gas analysis), and no withdrawal signs were precipitated by the administration of opioid (mu or delta) antagonists. Coupled with the previously published lack of self-administration behavior of the compound by alfentanil-trained primates, these findings strongly recommend delta-opioid agonists such as JNJ-20788560 for the relief of inflammatory hyperalgesia.

Top predators in the marine environment integrate chemical signals acquired from their prey that reflect both the species consumed and the regions from which the prey were taken. These chemical tracers-stable isotope ratios of carbon and nitrogen; persistent organic pollutant (POP) concentrations, patterns and ratios; and fatty acid profiles-were measured in blubber biopsy samples from North Pacific killer whales (Orcinus orca) (n=84) and were used to provide further insight into their diet, particularly for the offshore group, about which little dietary information is available. The offshore killer whales were shown to consume prey species that were distinctly different from those of sympatric resident and transient killer whales. In addition, it was confirmed that the offshores forage as far south as California. Thus, these results provide evidence that the offshores belong to a third killer whale ecotype. Resident killer whale populations showed a gradient in stable isotope profiles from west (central Aleutians) to east (Gulf of Alaska) that, in part, can be attributed to a shift from off-shelf to continental shelf-based prey. Finally, stable isotope ratio results, supported by field observations, showed that the diet in spring and summer of eastern Aleutian Island transient killer whales is apparently not composed exclusively of Steller sea lions.

Oil of mustard (OM) is a potent neuronal activator that promotes allodynia and hyperalgesia within minutes of application. In this study, OM was used to induce an acute colitis. We also investigated whether intracolonic OM-induced inflammation alters gastrointestinal (GI) function over a longer time frame as a model of postinflammatory irritable bowel syndrome (PI-IBS). Mice given a single administration of 0.5% OM developed a severe colitis that peaked at day 3, was reduced at day 7, and was absent by day 14. At the peak response, there was body weight loss, colon shrinkage, thickening and weight increases, distension of the proximal colon, and diarrhea. Macroscopic inspection of the distal colon revealed a discontinuous pattern of inflammatory damage and occasional transmural ulceration. Histological examination showed loss of epithelium, an inflammatory infiltrate, destruction of mucosal architecture, edema, and loss of circular smooth muscle architecture. OM administration increased transit of a carmine dye bolus from 58% of the total length of the upper GI tract in untreated age-matched controls to as high as 74% when tested at day 28 post-OM. Mice in the latter group demonstrated a significantly more sensitive response to inhibition of upper GI transit by the mu-opioid receptor agonist loperamide compared with normal mice. OM induces a rapid, acute, and transient colitis and, in the longer term, functional changes in motility that are observed when there is no gross inflammation and thereby is a model of functional bowel disorders that mimic aspects of PI-IBS in humans.

The enteric nervous system (ENS) is the division of the autonomic nervous system that regulates gastrointestinal (GI) function. Although large numbers of enteric neurons may be lost with age, the GI tract remains surprisingly functional. Exceptions to this generality include swallowing disorders and reduced colonic motility in the elderly. Evidence of age-related neurodegenerative changes in structure and function of the ENS is briefly reviewed in this Perspective.

Many individuals experience gastrointestinal (GI) dysfunction more frequently as they age, and the segment of the human population that is growing the most rapidly is the 'oldest old', who are >/= 80 years old. There has recently been renewed interest in the age-related changes intrinsic to the gut, and these investigations may help physicians understand the 'normal' aged GI tract, as distinct from disordered bowel function that is the result of comorbid conditions and/or GI side effects of medications used to treat those conditions. In this concise review we summarize recent data that suggest age-related neurodegenerative changes in the enteric nervous system (ENS) are key to functional changes observed with advanced age. Morphological studies are reviewed that demonstrate clearly the loss of enteric neurones in both submucosal and myenteric plexuses in humans and in rodents. Recent studies that indicate selective preservation of nitrergic, but not cholinergic, neurones are reviewed, as are preliminary findings that intrinsic sensory neurones may be among the most 'age-labile' subpopulations of the ENS. Caloric restriction remains the only intervention known that prevents neurodegeneration of ageing in the ENS, and mechanisms involved in this phenomenon are discussed. The field of ageing research in enteric neurobiology is ripe for rapid progression from phenomenology of age-related losses of neurones and associated functional changes to discovery of therapeutic approaches that may help ameliorate deterioration of bowel function and thereby contribute significantly to improved quality of life in advanced age.

Our understanding of the world around us and the many objects that we encounter is based primarily on three-dimensional information. It is simply part of the environment in which we live and the intuitive nature of our interpretation of our surroundings. In the arena of biomedical imaging, the image information most often collected is in the form of two-dimensional images. In cases where serial slice information is obtained, such as MRI images, it is still difficult for the observer to mentally build and understand the three-dimensional structure of the object. Although most image rendering software packages allow for 3D views of the serial sections, they lack the ability to segment, or isolated different objects in the data set. Typically the task of segmentation is performed by knowledgeable persons who tediously outline or label the object of interest in each image slice containing the object [1,2]. It remains a difficult challenge to train a computer to understand an image and aid in this process of segmentation. This article reports of on-going work in developing a semi-automated segmentation technique. The approach uses a Leica Confocal Laser Scanning Microscope (CLSM) to collect serial slice images, image rendering and manipulating software called IMOD (Boulder Colorado), and Matlab (The Mathworks Inc.) image processing tools for development of the object segmentation routines. The initial objects are simple fluorescent microspheres (Molecular Probes), which are easily imaged and segmented. The second objects are rat enteric neurons, which provide medium complexity in shape and size. Finally, the work will be applied to the biological cells of the household .y, Musca domestica, to further understand how its vision system operates.

As we enter the 21st century, the segment of the population that is the most rapidly expanding is that comprised of individuals 85 yr of age and older. Dysfunctions of the gastrointestinal (GI) system, including dysphagia, constipation, diarrhea, and irritable bowel syndrome are more common complaints of the elderly, yet our knowledge of the aging GI tract is incomplete. Compared with the rapid advances in the neurobiology of aging in the central nervous system, the understanding of age-related changes in the enteric nervous system (ENS) is poor. In this brief review, I recap experiments that reveal neurodegenerative changes and their functional correlates in the ENS of mice, rats, and guinea pigs. Clinical literature seems indicative of similar structural and functional age-related changes in the human ENS. Current studies that address the mechanisms underlying age-related changes in the ENS are introduced. The future directions for this field include physiological and pharmacological studies, especially at cellular and molecular levels. Research in the aging ENS is poised to make major advances, and this new knowledge will be useful for clinicians seeking to better understand and treat GI dysfunction in the elderly.

The development of melanocytes from neural crest-derived precursors that migrate along the dorsolateral pathway has been attributed to the selection of this route by cells that are fate-restricted to the melanocyte lineage. Alternatively, melanocytes could arise from nonspecified cells that develop in response to signals encountered while these cells migrate, or at their final destinations. In most animals, the bowel, which is colonized by crest-derived cells that migrate through the caudal branchial arches, contains no melanocytes; however, the enteric microenvironment does not prevent melanocytes from developing from crest-derived precursors placed experimentally into the bowel wall. To test the hypothesis that the branchial arches remove the melanogenic potential from the crest-derived population that colonizes the gut, the Silky fowl (in which the viscera are pigmented) was studied. Sources of crest included Silky fowl and quail vagal and truncal neural folds/tubes, which were cultured or explanted to chorioallantoic membranes alone or together with branchial arches or limb buds from Silky fowl, White Leghorn, or quail embryos. Crest and mesenchyme-derived cells were distinguished by using the quail nuclear marker. Melanocytes developed from Silky fowl and quail crest-derived cells. Melanocyte development from both sources was inhibited by quail and White Leghorn branchial arches (and limb buds), but melanocyte development was unaffected by branchial arch (and limb buds) from Silky fowl. These observations suggest that a factor(s) that is normally expressed in the branchial arches, and is lacking in animals with the Silky mutation, prevents cells with a melanogenic potential from colonizing the bowel., (Copyright 2002 Wiley-Liss, Inc.)

The morphological and physiological characteristics of myenteric neurons in the guinea pig distal colon were determined using Lucifer yellow- or N-(2-aminoethyl) biotinamide-containing microelectrodes and intracellular recording and staining methods. The neurons in this study (n = 204) were classified on the basis of the shapes of their cell bodies and short processes or dendrites and the number of long processes or axons as Dogiel type I (n = 75 neurons; 36.8%), filamentous (n = 31 neurons; 15.2%), Dogiel type II (n = 38 neurons; 18.6%), and unclassified (n = 60 neurons; 29.4%). All Dogiel type II neurons had action potentials followed by an after-spike hyperpolarization (AH), and most of them (84%) had large, smooth somata and filamentous, short processes in addition to multiple, long processes or axons. Most of Dogiel type I, filamentous, and unclassified neurons (98%) had a single, long process, but four Dogiel type I neurons and one unclassified neuron had two long processes terminating as varicosities within other ganglia or on the surface of longitudinal muscle. The projections of monoaxonal neurons were distributed equally between oral and aboral directions, and most of them received fast excitatory postsynaptic potentials (EPSPs). All of the Dogiel type II neurons and seven Dogiel type I neurons were positive for calbindin immunoreactivity, but three filamentous neurons received fEPSPs, had spikes followed by AH, and were negative for calbindin. The presence of calbindin-immunoreactive(-IR) neurons was quite variable among the ganglia. These results confirm that neither the presence of calbindin immunoreactivity nor the absence of fEPSPs can be used as a predictor of cellular morphology or electrophysiological properties of myenteric neurons in the distal colon., (Copyright 2001 Wiley-Liss, Inc.)

Studies of the guinea pig small intestine have suggested that serotonin (5-HT) may be a mucosal transmitter that stimulates sensory nerves and initiates peristaltic and secretory reflexes. We tested the hypothesis that guinea pig villus epithelial cells are able to inactivate 5-HT because they express the same 5-HT transporter as serotonergic neurons. A full-length cDNA, encoding a 630-amino acid protein (89.2% and 90% identical, respectively, to the rat and human 5-HT transporters) was cloned from the guinea pig intestinal mucosa. Evidence demonstrating that this cDNA encodes the guinea pig 5-HT transporter included 1) hybridization with a single species of mRNA ( approximately 3.7 kb) in Northern blots of the guinea pig brain stem and mucosa and 2) uptake of [3H]5-HT by transfected HeLa cells via a saturable, high-affinity (Michaelis constant 618 nM, maximum velocity 2.4 x 10(-17) mol . cell-1 . min-1), Na+-dependent mechanism that was inhibited by chlorimipramine > imipramine > fluoxetine > desipramine > zimelidine. Expression of the 5-HT transporter in guinea pig raphe and enteric neurons and the epithelium of the entire crypt-villus axis was demonstrated by in situ hybridization and immunocytochemistry. Inhibition of mucosal 5-HT uptake potentiates responses of submucosal neurons to mucosal stimulation. The epithelial reuptake of 5-HT thus appears to be responsible for terminating mucosal actions of 5-HT.

Megacolon occurs in neonatal and adult transgenic mice that overexpress the Hoxa-4 gene. Abnormalities, which are restricted to the terminal colon of these mice, include a hypoganglionosis, abnormal enteric ganglia with a structure appropriate for extra-enteric peripheral nerve and not the enteric nervous system (ENS), and gaps in the longitudinal muscle occupied by ganglia. To investigate the developmental origin of these abnormalities, we analyzed the development of the pelvis and terminal colon in Hoxa-4 transgenic mice. Morphological abnormalities were detected as early as E13. These included an enlargement of the mucosa and the bowel wall, a thickening of the enteric mesenchyme, and the ectopic location of pelvic ganglion cells, which initially clustered on the dorsolateral wall of the hindgut. As the bowel enlarged, these ectopic cells become ventrolateral and, between days E17 and E18.5, appeared to become incorporated into the gut, leaving neuron-filled gaps in the longitudinal muscle layer. The ectopic ganglia retained extra-enteric characteristics, including the presence of capillaries, basal laminae, collagen fibers, and catecholaminergic neurons, even after their incorporation into the bowel. It is proposed that the abnormal and ectopic expression of the Hoxa-4 transgene in the colon causes signalling molecule(s) of the enteric mesenchyme to be overproduced and that the overabundance of these signals leads to mucosal enlargement and misdirection of migrating pelvic neuronal progenitors.

The roles of 5-hydroxytryptamine3 (5-HT3), 5-HT4, and nicotinic receptors in the peristaltic reflex were investigated in isolated segments of guinea pig distal colon. The reflex assessed by measuring the propulsion of solid pellets, was affected neither by 5-HT3-selective antagonists (ondansetron granisetron) nor by 5-HT4-selective antagonists (SDZ-205-557, GR-113808A, SB-204070) applied individually (1.0 microM); nevertheless, the reflex was inhibited by combining these antagonists or by applying a 5-HT3/5-HT4 dual antagonist (FK-1052). Hexamethonium abolished the peristaltic reflex at 100 microM, but not at 10-32 microM. In contrast, the peristaltic reflex was inhibited when hexamethonium (32 microM was combined with either a 5-HT3- or 5-HT4-selective antagonist (1.0 microM). These observations suggest that 5-HT3, 5-HT4, and nicotinic receptors participate in the initiation and/or propagation of the peristaltic reflex. The data are consistent with the idea that these receptors are arranged in parallel in the neural pathways that mediate the peristaltic reflex in the distal colon.

Background: Subsets of myenteric neurons have been identified. To determine the proportional representation of neurons in each, it is necessary to relate the number of neurons in the subset to that of the complete set. Prior estimates of total numbers of neurons, obtained with many different markers, have varied widely., Methods: Markers were compared for counting myenteric neurons in dissected laminar preparations of guinea pig duodenum, jejunum-ileum, and colon; the effect of stretching preparations on these counts was also determined. Markers included the visualization of single-stranded nucleic acid with cuprolinic blue and the immunocytochemical demonstration of neuron specific enolase (NSE), PGP9.5, S-100, and the constitutive expression of a Fos related antigen (FRA)., Results: Neurons could not be counted accurately by demonstrating NSE, PGP9.5, or S-100. The number of neurons detected by demonstrating FRA was consistently less than that determined with cuprolinic blue (approximately 65%). Cuprolinic blue-derived estimates of neuron numbers were higher than most reported in the literature, but comparable to those recently obtained with "a nerve cell body" antiserum. Ganglionic area was found to be stretch independent. The rank order of neurons/cm2 and ganglionic area/ unit resting length was colon > duodenum >> jejunum-ileum; more neurons were found in the myenteric plexus of the colon (7.3 x 10(6)) than in that of the entire small intestine (6.5 x 10(6))., Conclusions: Prior studies that have obtained denominators for estimating the proportions of myenteric neuronal subsets with markers that do not reveal the entire population should be re-evaluated. The guinea pig colon contains a surprisingly large number of neurons, the physiological significance of which must be determined.

A 2-year-old spayed female Siamese cat was presented with clinical liver disease characterized by anorexia; depression; elevations in serum levels of alanine aminotransferase, aspartate aminotransferase, and lactate dehydrogenase; hyperbilirubinemia; and icterus. Liver biopsy diagnosed hepatocellular degeneration with marked centrilobular hepatocellular accumulation of rhodanine-positive brown granules. Subsequent postmortem examination revealed similar granular material in the epithelium of the proximal convoluted tubules and collecting ducts of the kidney and alveolar epithelium and macrophages in the lung. The liver and kidney copper concentrations were 4,074 and 792 ppm dry weight, respectively. Hepatic degeneration in this cat apparently was due to excessive accumulation of copper.

The effects of anti-idiotypic antibodies (alpha-id) that recognize serotonin [5-hydroxytryptamine (5-HT)] receptors on myenteric neurons of the guinea pig small intestine were characterized electrophysiologically, and alpha-id binding sites were located immunocytochemically. Initial applications of the alpha-id mimicked each of three actions of 5-HT: a rapid depolarization, associated with a fall in input resistance (Rin), which was inhibited by the 5-HT3 antagonists tropisetron (> or = 1 microM) and renzapride (100 microM); a slow membrane depolarization, associated with increased Rin, that was inhibited by the 5-HT1P antagonist renzapride but was unaffected by a 5-HT4 blocking concentration of tropisetron (10 microM); and a hyperpolarization, associated with decreased Rin, that was antagonized by the 5-HT1A inhibitor NAN-190. Cross-desensitization was observed between responses to 5-HT and the alpha-id. After exposure to the alpha-id, subsequent responses to the alpha-id, 5-HT, and stimulus-evoked slow excitatory postsynaptic potentials were antagonized; however, responses to carbachol and substance P were unaffected. The alpha-id thus specifically inhibits the effects of endogenously released and exogenously applied 5-HT. The alpha-id bound to sites on myenteric and submucosal neurons and a subepithelial nerve plexus. Binding of the alpha-id was blocked by 5-HT1P-, 5-HT3-, and 5-HT4-specific antagonists. We concluded that the alpha-id binds selectively to all known subtypes of 5-HT receptor in the enteric nervous system and is thus useful for investigating the gastrointestinal function of 5-HT.

Serotonin (5-HT) is a mediator (through 5-HT1P receptors) of slow EPSPs in myenteric ganglia of the small intestine. The effect of 5-HT can be mimicked by elevating cAMP; therefore, we tested the hypothesis that the slow EPSP-like response to 5-HT is cAMP-mediated. Guinea pig gut was enzymatically dissociated; myenteric ganglia remained intact and were collected by filtration. Neurons in the isolated ganglia retained their ability to manifest the slow EPSP-like response to 5-HT. Exposure to 5-HT raised the ganglionic level of cAMP (ED50 0.3 microM). This effect was not antagonized by the 5-HT1P antagonist, N-acetyl-5-hydroxytryptophyl-5-hydroxytryptophan amide (100.0 microM), or mimicked by the 5-HT1P agonist, 5-hydroxyindalpine (10.0 microM). Increases in cAMP were also evoked by the 5-HT1 agonist, 5-carboxyamidotryptamine (10.0 microM), the 5-HT2 agonist, (+-)-1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI; 1.0-10.0 microM), and by the 5-HT4 agonists, renzapride (1.0-10.0 microM) and 5-methoxytryptamine (1.0-10.0 microM); however, neither the 5-HT1/5-HT2 antagonists, spiperone, methysergide, and methiothepin, nor the 5-HT4 antagonist, tropisetron (ICS 205-930; 10.0 microM), were able to inhibit the rise in cAMP evoked by these compounds or by 5-HT (0.1-10.0 microM). The 5-HT-evoked elevation of cAMP was antagonized by ketanserin (10.0 microM), which also blocked the effects of 5-methoxytryptamine and DOI, but not those of renzapride. The effective concentration of DOI, however, was higher than that needed for activation of 5-HT2 receptors, and Northern analysis using a cDNA probe encoding the rat 5-HT2 receptor failed to reveal the presence of 5-HT2 mRNA in myenteric ganglia, although it hybridizes with mRNA of the right size in the guinea pig brain. Compounds that failed to change levels of cAMP or to antagonize the action of 5-HT included 8-hydroxy-di-n-propylamino tetralin, R58639, R88226, and sumatriptan. It is concluded that the receptor responsible for the 5-HT-induced rise in cAMP in ganglia isolated from the guinea pig myenteric plexus is not a known subtype of 5-HT receptor. Since the pharmacology of this novel receptor is different from that of the slow EPSP-like response to 5-HT, the receptor probably does not mediate the slow EPSP.

Two subtypes of excitatory 5-hydroxytryptamine (5-HT) receptor, 5-HT1P and 5-HT3, are found on type 2-AH neurons of the guinea pig myenteric plexus. The 5-HT1P receptor mediates a slow and the 5-HT3 receptor a fast depolarization of these cells, however, the role of these receptors in the physiology of the gut is unknown. Renzapride (BRL 24924), a substituted benzamide, has previously been found to antagonize responses of myenteric neurons mediated by both 5-HT1P and 5-HT3 receptors. The effects on myenteric type 2-AH neurons of a structurally similar benzamide, zacopride, which unlike renzapride has S and R stereoisomers, were investigated to gain further insight into 5-HT receptor function. In contrast to renzapride, S-, but not R-zacopride, was found to mimic the 5-HT1P receptor-mediated slow response to 5-HT. Desensitization of 5-HT1P receptors with 5-HT inhibited slow depolarizing responses to S-zacopride, and desensitization with S-zacopride antagonized slow responses to 5-HT. Responses to S-zacopride were also inhibited by renzapride and the 5-HT1P receptor antagonist N-acetyl-5-hydroxytryptophyl-5-hydroxytryptophan amide (5-HTP-DP). S-zacopride, like renzapride and 5-HT, presynaptically inhibited nicotinic fast excitatory postsynaptic potentials, an effect that can be mediated by 5-HT1P or 5-HT1A receptors. Both S and R stereoisomers of zacopride antagonized 5-HT3 receptor-mediated fast responses to 5-HT. Unlike 5-HTP-DP, neither zacopride or its stereoisomers nor renzapride inhibited the binding of 5-[3H]HT to 5-HT1P receptors. [3H]zacopride (5-10 nM) was found to bind to a site in the gut from which it could be displaced by a 1,000-fold excess of renzapride and S-zacopride (but not R-zacopride) greater than 5-HTP-DP much greater than the 5-HT3 receptor antagonist ICS 205-930. These observations suggest that, in addition to 5-HT3 receptors, there is a benzamide binding site on myenteric neurons that interacts with, but is distinct from, the 5-HT recognition site of 5-HT1P receptors. Benzamides may affect coupling of the 5-HT1P receptor to its effector.

5-Hydroxytryptamine (5-HT) receptors have been analyzed and related to potential roles played by 5-HT in the physiology of the enteric nervous system (ENS). Three subtypes of 5-HT receptor--5-HT1P, 5-HT3, and 5-HT1A--have been found on enteric neurons. Receptors have been identified by intracellularly recording the electrical activity of enteric neurons and by studying the binding of radioligands and polyclonal anti-idiotypic antibodies raised against antibodies to 5-HT. Radioligand binding has been assessed by rapid filtration and by radioautography. 5-HT1P receptors mediate slow depolarizations of myenteric neurons that are associated with a closure of K+ channels. These responses can be inhibited by N-acetyl-hydroxytryptophyl-5-hydroxytryptophan amide (5-HTP-DP) and by the substituted benzamide, BRL 24924. 5-HT1P-like responses can be mimicked by 5- and 6-hydroxyindalpine, by another substituted benzamide, the S stereoisomer of zacopride, and by anti-idiotypic antibodies. 5-HT1P receptors can be labeled by 3H-5-HT and 3H-5-hydroxyindalpine with high affinity and are located on neurons of both enteric plexuses and on processes of intrinsic neurons in the gastrointestinal mucosa. A similar distribution of binding sites for anti-idiotypic antibodies is revealed by immunocytochemistry. Excitatory postsynaptic potentials (EPSPs) mediated by 5-HT are abolished by 5-HT1P antagonists. Blockade of 5-HT1P receptors is accompanied by acceleration of the rate of gastric emptying. Mucosal application of cholera toxin activates enteric neurons in both plexuses; this action is blocked by 5-HT1P or 5-HT3 antagonists and by anti-idiotypic antibodies. 5-HT3 receptors are responsible for fast depolarizations associated with increased membrane conductance. These responses are antagonized by ICS 205-930 and mimicked by 2-methyl-5-HT and anti-idiotypic antibodies. 5-HT1A receptors have been reported to mediate hyperpolarizing responses associated with a rise in membrane conductance. Hyperpolarizing responses are also elicited by the 5-HT1A agonists, 8-hydroxy-di-n-propylaminotetralin (8-OH-DPAT) and 5-carboxyamidotryptamine. It is proposed that 5-HT1P receptors and perhaps 5-HT3 receptors are involved in initiating the peristaltic reflex and in regulating gastric emptying. No physiologic role has yet been identified for 5-HT1A receptors in the ENS.

Enterochromaffin cells of adult mouse duodenum were studied with light- and electron-microscopical techniques. They were distinguished from other enteroendocrine cells by their pleomorphic, electron-dense secretory granules in the basal cytoplasm. At the apices of enterochromaffin cells, tufts of short microvilli bordered the gut lumen. At their bases, irregular cytoplasmic extensions were either in contact with or passed through the basal lamina. The presence of cytoplasmic extensions in close proximity to fenestrated capillaries and subepithelial nerves suggested an endocrine or paracrine function. Electron micrographs of serial thin sections were used to reconstruct an enterochromaffin cell from the crypt epithelium in three dimensions and to determine its relationship with the underlying neural plexus. Although extensions from the serially sectioned and reconstructed cell and other enterochromaffin cells studied in crypt epithelia protruded through the basal lamina, no synaptic contacts were seen. Evidence of a synaptic contact between a neurite and another type of enteroendocrine cell (possibly an intestinal A cell), suggested a neurocrine role for some of the basally-granulated cells. Possible functions of enterochromaffin cells are discussed in the light of recent literature on the system of enteroendocrine cells, also known as APUD (amine precursor uptake and decarboxylation) cells and/or paraneurons.

Intracellular recording methods were used in vitro to analyze the synaptic behavior of neurons in myenteric ganglia of guinea pig distal colon. Fast excitatory postsynaptic potentials (EPSPs) were observed in a variety of types of colonic neurons. Both spontaneous and stimulus-evoked EPSPs were abolished or suppressed by addition of hexamethonium, tetrodotoxin, or elevation of Mg2+ and reduction of Ca2+ in the bathing medium. Individual neurons usually received inputs from several fiber tracts and multiple EPSPs were sometimes evoked by electrical stimulation of single-fiber tracts. Stimulus-evoked fast EPSPs were always of greater amplitude, longer duration, and longer decay time than were spontaneous fast EPSPs in the same neurons. No rundown of the fast EPSPs occurred during prolonged stimulation at frequencies up to 10 Hz. Repetitive stimulation evoked slow depolarizing potentials (slow EPSPs) in 25% of the neurons. Characteristics of the slow EPSPs were 1) slow rise times, 2) duration in the seconds time domain, 3) enhanced excitability, 4) increased input resistance, and 5) reduction of hyperpolarizing after-potentials. In general, the variety of synaptic potentials and the properties of the events were the same as found in myenteric neurons of the guinea pig small bowel. Compared with synaptic behavior of small intestinal myenteric neurons, the notable differences were absence of the rundown phenomenon for fast EPSPs in the colonic neurons and a greater incidence of spontaneously occurring fast EPSPs.

Intracellular recording was used in vitro to analyze electrophysiological properties of neurons in myenteric ganglia of guinea pig distal colon. The neurons were classified into six types based on their electrical behavior. Type 1 colonic neurons discharged action potentials throughout depolarizing current pulses and were otherwise similar to S/type 1 neurons found in the guinea pig small bowel. The second type had passive and active electrical properties similar to those of AH/type 2 myenteric neurons of the small intestine. These cells discharged only a single spike at the onset of depolarizing current pulses, and the spikes were followed by long-lasting hyperpolarizing afterpotentials. Excitability of the type 2 neurons was enhanced in the presence of elevated Mg2+ and reduced Ca2+, and the spikes were unaffected by tetrodotoxin. Type 3 colonic neurons showed fast synaptic potentials but did not generate action potentials. The majority of neurons were referred to as type 2 colonic neurons. Type 4 neurons discharged single action potentials only at the onset of depolarizing current pulses, and the spikes were not followed by prolonged hyperpolarizing afterpotentials. Unlike type 2 neurons, excitability remained unchanged in the presence of reduced extracellular Ca2+ and elevated Mg2+. Action potentials of type 4 neurons were suppressed or abolished by tetrodotoxin. A group of spontaneously active neurons was classified as type 5 colonic neurons. Type 6 cells were inexcitable and assumed to be glial cells.

Intracellular methods were used to study the effects of serotonin and substance P (SP) on the electrical behavior of myenteric neurons in guinea-pig distal colon in vitro. Serotonin evoked either a short-duration transient depolarization, a long-lasting depolarization or a multiphasic response consisting of a rapid depolarization followed by a short duration hyperpolarizing potential and then a long-lasting depolarization. Application of SP evoked a long-lasting depolarization. Depolarizing potentials to both substances were accompanied by enhanced excitability that was reflected by repetitive spike discharge. Long-lasting depolarizations were associated with increased input resistance. The responses to serotonin or SP were unaltered by the presence of tetrodotoxin, hexamethonium or elevated extracellular Mg2+ and reduced Ca2+. Some neurons responded to both serotonin and SP indicating that both receptors coexisted on the same neuron. The putative SP antagonist, [D-Arg1,D-Phe5,D-Trp7,9,Leu11]SP did not affect the responses to SP. It did suppress the slow-depolarizing response to serotonin, while the fast response was unaffected. The responses to serotonin and SP in myenteric neurons of guinea-pig colon resembled the responses reported by others for small intestinal myenteric neurons.