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The English spotting coat color locus in rabbits, also known as Dominant white spotting locus, is determined by an incompletely dominant allele (En). Rabbits homozygous for the recessive wild-type allele (en/en) are self-colored, heterozygous En/en rabbits are normally spotted, and homozygous En/En animals are almost completely white. Compared to vital en/en and En/en rabbits, En/En animals are subvital because of a dilated (“mega”) cecum and ascending colon. In this study, we investigated the role of the KIT gene as a candidate for the English spotting locus in Checkered Giant rabbits and characterized the abnormalities affecting enteric neurons and c-kit positive interstitial cells of Cajal (ICC) in the megacolon of En/En rabbits. Twenty-one litters were obtained by crossing three Checkered Giant bucks (En/en) with nine Checkered Giant (En/en) and two en/en does, producing a total of 138 F1 and backcrossed rabbits. Resequencing all coding exons and portions of non-coding regions of the KIT gene in 28 rabbits of different breeds identified 98 polymorphisms. A single nucleotide polymorphism genotyped in all F1 families showed complete cosegregation with the English spotting coat color phenotype (θ = 0.00 LOD  = 75.56). KIT gene expression in cecum and colon specimens of En/En (pathological) rabbits was 5–10% of that of en/en (control) rabbits. En/En rabbits showed reduced and altered c-kit immunolabelled ICC compared to en/en controls. Morphometric data on whole mounts of the ascending colon showed a significant decrease of HuC/D (P<0.05) and substance P (P<0.01) immunoreactive neurons in En/En vs. en/en. Electron microscopy analysis showed neuronal and ICC abnormalities in En/En tissues. The En/En rabbit model shows neuro-ICC changes reminiscent of the human non-aganglionic megacolon. This rabbit model may provide a better understanding of the molecular abnormalities underlying conditions associated with non-aganglionic megacolon. [ABSTRACT FROM AUTHOR]

In vertebrates, chemosensitivity of nutrients occurs through the activation of taste receptors coupled with G-protein subunits, including α-transducin (G αtran) and α-gustducin (G αgust). This study was aimed at characterising the cells expressing G αtran immunoreactivity throughout the mucosa of the sea bass gastrointestinal tract. G αtran immunoreactive cells were mainly found in the stomach, and a lower number of immunopositive cells were detected in the intestine. Some G αtran immunoreactive cells in the stomach contained G αgust immunoreactivity. Gastric G αtran immunoreactive cells co-expressed ghrelin, obestatin and 5-hydroxytryptamine immunoreactivity. In contrast, G αtran immunopositive cells did not contain somatostatin, gastrin/cholecystokinin, glucagon-like peptide-1, substance P or calcitonin gene-related peptide immunoreactivity in any investigated segments of the sea bass gastrointestinal tract. Specificity of G αtran and G αgust antisera was determined by Western blot analysis, which identified two bands at the theoretical molecular weight of ~45 and ~40 kDa, respectively, in sea bass gut tissue as well as in positive tissue, and by immunoblocking with the respective peptide, which prevented immunostaining. The results of the present study provide a molecular and morphological basis for a role of taste-related molecules in chemosensing in the sea bass gastrointestinal tract. [ABSTRACT FROM AUTHOR]

Spinal ganglia (SG) neurons are commonly classified according to various specific features. The most widespread classification based on morphological and ultrastructural features subdivides SG neurons into light and small dark neurons. Using immunohistochemical, histochemical and lectin methods, it is possible to further subdivide the small dark neurons into two subpopulations: peptidergic and nonpeptidergic neurons. The majority of studies on SG neurons were carried out on mice and rats; there are few or no studies on large mammals. In this study, some of the widely used neuronal markers, neurofilament 200 kDa (NF200), substance P (SP), calcitonin gene-related peptide (CGRP) and isolectin B4 (IB4), were employed to characterize neuronal nitric oxide synthase (nNOS)-immunoreactivity (-IR) in sheep ( Ovis aries) SG (Th13-L2) neurons. The majority of the SG neurons were IB4-labeled (79 ± 10%), followed by NF200- (45 ± 4%), NOS- (44 ± 10%), SP- (42 ± 5%) and CGRP-IR (35 ± 7%) neurons. The triple staining experiments showed that a higher percentage (75 ± 16%) of NOS-IR neurons bound both IB4 and CGRP, or both IB4 and SP (49 ± 6%). The IB4 marker showed an unexpected staining pattern; in fact, IB4-labeled neurons largely colocalized with NF200, usually considered a marker of light SG neurons, and with CGRP and SP. For this reason, IB4 cannot be employed in sheep to differentiate between light and dark neurons, or between peptidergic and nonpeptidergic neurons. These results suggest the importance of being cautious when comparing data among different species. Microsc. Res. Tech., 2010. © 2009 Wiley-Liss, Inc. [ABSTRACT FROM AUTHOR]

Background: Adenosine affects the tone and reactivity of airways by activating specific membrane receptors, named A1, A2a, A2b and A3. It affects cellular activities either directly by regulating membrane ion exchanges and polarization, or indirectly by modifying neurotransmitter release. Objectives: We assessed the effect of A1 and A3 receptor activation on electrically induced nonadrenergic, noncholinergic (NANC) relaxations in the guinea pig isolated trachea and the localization of A1 and A3 receptors in tracheal inhibitory neurons. Methods: NANC responses at 3 Hz were evaluat- ed in the presence of 2-chloro-N6-cyclopentyladenosine (CCPA), a selective A1 agonist, and 2-chloro-N6-(3-iodobenzyl)-adenosine-5′-N-methyluronamide (Cl-IB-MECA), a selective A3 agonist, before and after the administration of 8-cyclopentyl-1,3-dipropylxanthine (DPCPX), a selective A1 antagonist, or 9-chloro-2-(2-furanyl)-5-((phenylacetyl)amino[1,2,4]triazolo[1,5-c])quinazoline (MRS 1220), a selective A3 antagonist, respectively. For immunohistochemistry, tissues were exposed to antibodies to HuC/D, a general neuronal marker, neuronal nitric oxide synthase (nNOS), and A1 or A3 adenosine receptors and processed by indirect immunofluorescence. Results: CCPA (10 nM–3 μM) inhibited NANC relaxations. DPCPX (10 nM) failed to antagonize the effect of CCPA, but inhibited per se NANC relaxations (range 0.1–100 nM). CCPA (10 nM–10 μM) contracted unstimulated tracheal preparations, an effect antagonized by 10 nM DPCPX, with a pKB value of 8.43. Cl-IB-MECA (10 nM–3 μM) inhibited NANC relaxations through a mechanism antagonized by MRS 1220 (100 nM). A1- and A3-positive neurons containing nNOS were detected in tracheal sections. Conclusions: Enogenous adenosine may induce airway hyperresponsiveness by inhibiting NANC relaxations via A1 and A3 receptors. Copyright © 2008 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Bacterial single cell protein (SCP) is considered a promising circular protein ingredient for aquafeed, due to the high protein content, and for the possibilities to grow them on different substrates such as organic waste thus leading to low environmental footprint and affordable production costs. Their use as raw material has been assessed in several farmed species, however, research on Mediterranean ones is still scarce. Hence, a study was undertaken to evaluate growth, plasma biochemistry, gut histology and gut microbiota (GM) response of gilthead sea bream (Sparus aurata) fed diets with increasing levels of bacterial SCP in comparison to a control without SCP. Three isonitrogenous and isolipidic extruded diets (44% protein; 19% lipid) were formulated with different bacterial SCP (derived from Corinebacterium glutamicum) level (10% SCP, SPC10; 15% SCP, SCP15; 20% SCP, SCP20) to replace vegetable protein ingredients (total replacement of soy protein concentrate and partial replacement of corn gluten), while a control diet (CTRL) was formulated without SCP. Fish groups of 45 individuals (initial weight: 75 g) were fed to visual satiation over 108 days. At the end of the trial there were no significant differences on growth, feed intake, feed conversion rate, protein efficiency ratio and protein apparent digestibility. Most of plasma parameters were found to be equal for all treatments, except for those related to nucleic acids molecules degradation such as phosphorus and urea which were higher in SCP10 and SCP20 compared to CTRL, respectively. No morphological alterations were found in the intestines of any fish analysed. Different responses of the overall GM structure in relation to the bacterial SCP inclusion level were detected. Specifically, SCP exerted a positive effect on GM internal diversity which increased at increasing dietary SCP inclusion level. In addition, SCP inclusion lead to increase in the abundance of Bacillus spp., taxa which can potentially support nutrition, immune system, and disease resistance. In conclusion, it seems feasible to include up to 20% of SCP dietary level for gilthead sea bream without compromising growth, feed efficiency and health parameters. • SCP from Corinebacterium glutamicum was included up to 20% in seabream diet. • SCP did not affect growth, and feed efficiency when replacing vegetable protein. • SCP at each level tested, determined a shift in the gut microbiota structure. • SCP promoted beneficial taxa (Bacillus spp.) in the gut microbiota. [ABSTRACT FROM AUTHOR]

Bitter taste receptors (T2Rs) are expressed in the mammalian gastrointestinal mucosa. In the mouse colon, T2R138 is localized to enteroendocrine cells and is upregulated by long-term high fat diet that induces obesity. The aims of this study were to test whether T2R38 expression is altered in overweight/obese (OW/OB) compared to normal weight (NW) subjects and characterize the cell types expressing T2R38, the human counterpart of mouse T2R138, in human colon. Colonic mucosal biopsies were obtained during colonoscopy from 35 healthy subjects (20 OW/OB and 15 NW) and processed for quantitative RT-PCR and immunohistochemistry using antibodies to T2R38, chromogranin A (CgA), glucagon like peptide-1 (GLP-1), cholecystokinin (CCK), or peptide YY (PYY). T2R38 mRNA levels in the colonic mucosa of OW/OB were increased (> 2 fold) compared to NW subjects but did not reach statistical significance (P = 0.06). However, the number of T2R38 immunoreactive (IR) cells was significantly increased in OW/OB vs. NW subjects (P = 0.01) and was significantly correlated with BMI values (r = 0.7557; P = 0.001). In both OW/OB and NW individuals, all T2R38-IR cells contained CgA-IR supporting they are enteroendocrine. In both groups, T2R38-IR colocalized with CCK-, GLP1- or PYY-IR. The overall CgA-IR cell population was comparable in OW/OB and NW individuals. This study shows that T2R38 is expressed in distinct populations of enteroendocrine cells in the human colonic mucosa and supports T2R38 upregulation in OW/OB subjects. T2R38 might mediate host functional responses to increased energy balance and intraluminal changes occurring in obesity, which could involve peptide release from enteroendocrine cells. [ABSTRACT FROM AUTHOR]

The objective of the present study was to verify if polyurethane foam is a suitable material to make accurate casts of vessels and viscera, and to develop a method based on its use for anatomical studies. This new technique has been tested primarily on the lungs of different animals, but also on the renal, intestinal and equine digital vessels. It consisted of three steps: specimen preparation, injection of the foam and corrosion of the cast. All structures injected with foam were properly filled. The bronchial tree and the vessels could be observed up to their finer branches. The method is inexpensive, simple and requires no special equipment. The pre-casting procedure does not require perfusion of the specimens with formalin, or prolonged flushing with carbon dioxide gas or air for drying. The polyurethane foam does not need a catalyst. It is simply diluted with acetone, which does not cause shrinkage of the cast due to evaporation during hardening. The foam naturally expands into the cavities without high pressure of the inoculum, and hardens in just 2 or 3 h at room temperature. Only two drawbacks were observed. The first is the fact that multiple injections cannot be made in the same cavity since the foam solidifies quickly; the second is the slight brittleness of the cast, due to the low elasticity of polyurethane foam. In conclusion, polyurethane foam was a suitable material for producing accurate casts of vessels and viscera. [ABSTRACT FROM AUTHOR]

The English spotting coat color locus in rabbits, also known as Dominant white spotting locus, is determined by an incompletely dominant allele (En). Rabbits homozygous for the recessive wild-type allele (en/en) are self-colored, heterozygous En/en rabbits are normally spotted, and homozygous En/En animals are almost completely white. Compared to vital en/en and En/en rabbits, En/En animals are subvital because of a dilated (“mega”) cecum and ascending colon. In this study, we investigated the role of the KIT gene as a candidate for the English spotting locus in Checkered Giant rabbits and characterized the abnormalities affecting enteric neurons and c-kit positive interstitial cells of Cajal (ICC) in the megacolon of En/En rabbits. Twenty-one litters were obtained by crossing three Checkered Giant bucks (En/en) with nine Checkered Giant (En/en) and two en/en does, producing a total of 138 F1 and backcrossed rabbits. Resequencing all coding exons and portions of non-coding regions of the KIT gene in 28 rabbits of different breeds identified 98 polymorphisms. A single nucleotide polymorphism genotyped in all F1 families showed complete cosegregation with the English spotting coat color phenotype (θ = 0.00 LOD  = 75.56). KIT gene expression in cecum and colon specimens of En/En (pathological) rabbits was 5–10% of that of en/en (control) rabbits. En/En rabbits showed reduced and altered c-kit immunolabelled ICC compared to en/en controls. Morphometric data on whole mounts of the ascending colon showed a significant decrease of HuC/D (P<0.05) and substance P (P<0.01) immunoreactive neurons in En/En vs. en/en. Electron microscopy analysis showed neuronal and ICC abnormalities in En/En tissues. The En/En rabbit model shows neuro-ICC changes reminiscent of the human non-aganglionic megacolon. This rabbit model may provide a better understanding of the molecular abnormalities underlying conditions associated with non-aganglionic megacolon. [ABSTRACT FROM AUTHOR]

R4047 --> 612.1 --> The enteric nervous system (ENS), the neural network that controls gastrointestinal functions, varies among species. This is particularly evident for the submucosal plexus that is multilayered in humans but single layered and much less developed in rodents and guinea pigs often used for ENS studies. The submucosal plexus plays a major role in the control of gut functions. The pig has many homologies with humans, including a well‐developed submucosal plexus with an inner submucous plexus (ISP) near the mucosa and an outer submucous plexus (OSP) near the circularmuscle, as in humans. We have previously shown that in the adult pig colon, the density of enteric neurons is significantly higher in the ISP than OSP in ascending (AC) and descending (DC) colon, and that neurons in both plexuses contain choline acetyltransferase (ChAT) immunoreactivity (IR) and neuronal nitric oxide synthase (nNOS)‐IR, markers of the major excitatory (acetylcholine) and inhibitory (nitric oxide) transmitters with a small subpopulation containing both transmitters (Cell Tissue Res. 2021;383(2):645‐654). In this study, we compared the density and neurochemical profile of enteric neurons of the ISP and OSP in the AC and DC of the adult pig (16 Yucatan pigs, 3 F,25‐30 kg) and humans. Human specimens were obtained from 14 patients (6 F, age 48‐86) undergoing elective surgery for colon adenocarcinoma and were taken from the uninvolved resection margin. We used HuC/D (pan‐neuronal marker), ChAT, nNOS and substance P (SP) antibodies in whole mount preparations of the submucosa, confocal imaging and Imaris software for quantification. In both pig and human colon, the highest density of neurons was observed in the ISP of the AC, though the overall density of HuC/D‐IR neurons was about 4 times higher in pig vs. human. As observed in the pig colon, the density of HuC/D‐IR neurons in the ISP of the AC was significantly higher than the ISP of the DC (P<0.01), whereas neuronal density in OSP of AC and DC were not significantly different. The ganglia were also smaller in human ISP and OSP throughout the colon (average ranging from 13‐28 neurons/ganglion) compared to pig where average neurons/ganglion ranged from 30‐120. The % of ChAT‐IR neurons in human (38‐44%) and pig (30‐44%) colon was comparable with no differences between ISP and OSP in AC and DC. The % of nNOS‐IR neurons in human colon was similar in the ISP and OSP of AC and DC (16‐22%), which differs from the pig, where the % of nNOS‐IR neurons in the ISP of AC was significantly lower than OSP (15% vs. 45%) and both ISP and OSP of DC (38‐42%). Overall, the density of nNOS‐IR neurons in human colon was lower compared to the pig colon. The % of SP‐IR neurons was slightly lower in human (15‐20%) vs. pig (23‐26%) colon in both plexuses in the AC and DC. There were dense SP‐IR varicosities through the ganglia in both pig and human colon. SP‐IR mostly colocalized with ChAT‐IR but was also observed in a subpopulation of nNOS‐IR and of nNOS/ChAT‐IR neurons in both human and pig colon. Overall, except for a few differences in neuronal density, there are neurochemical similarities that support the validity of the pig as pre‐clinical model for translational research to explore neuromodulation in colonic disorders in humans. [ABSTRACT FROM AUTHOR]

The activity of the gastrointestinal tract is regulated through the activation of adrenergic receptors (ARs). Since data concerning the distribution of ARs in the horse intestine is virtually absent, we investigated the distribution of β2-AR in the horse ileum using double-immunofluorescence. The β2-AR-immunoreactivity (IR) was observed in most (95%) neurons located in submucosal plexus (SMP) and in few (8%) neurons of the myenteric plexus (MP). Tyrosine hydroxylase (TH)-IR fibers were observed close to neurons expressing β2-AR-IR. Since β2-AR is virtually expressed in most neurons located in the horse SMP and in a lower percentage of neurons in the MP, it is reasonable to retain that this adrenergic receptor could regulate the activity of both secretomotor neurons and motor neurons innervating muscle layers and blood vessels. The high density of TH-IR fibers near β2-AR-IR enteric neurons indicates that the excitability of these cells could be directly modulated by the sympathetic system. [ABSTRACT FROM AUTHOR]

We have examined the innervation of the gut-associated lymphoid system of the sheep ileum, with a view to identifying potential sites for neuroinvasion by pathogens, such as prions (PrPSc). Special attention has been paid to the follicles of Peyer’s patches (PPs), which are major sites of PrPSc accumulation during infection. Evidence exists that the enteric nervous system, together with the parasympathetic and sympathetic pathways projecting to the intestine, are important for PrPSc entry into the central nervous system. Thus, PrPSc might move from PPs to the neurons and nerve fibres that innervate them. We investigated, by immunohistochemistry and retrograde tracing (DiI) from the follicles, the distribution and phenotype of enteric neurons innervating the follicles. Antibodies against protein gene product 9.5, tyrosine hydroxylase, dopamine β hydroxylase, choline acetyltransferase, calbindin (CALB), calcitonin gene-related peptide (CGRP), and nitric oxide synthase were used to characterise the neurons. Immunoreactivity for each of these was observed in fibres around and inside PP follicles. CGRP-immunoreactive fibres were mainly seen at the follicular dome. Retrograde tracing revealed submucosal neurons that contributed to the innervation of PPs, including Dogiel type II neurons and neurons immunoreactive for CALB and CGRP. The major source of the adrenergic fibres are the sympathetic ganglia. Our results thus suggest that enteric and sympathetic neurons are involved during the first stage of neuroinvasion, with neurons connecting to them acting as potential carriers of PrPSc to the central nervous system. [ABSTRACT FROM AUTHOR]

We have identified the enteric neuron types expressing immunoreactivity for the calcium-binding protein calbindin D28k (CALB) in cryostat sections and whole-mount preparations of myenteric (MP) and submucosal (SMP) plexuses of sheep ileum. We wished to determine whether CALB-IR in the sheep enteric nervous system was expressed in Dogiel type II cells, as in guinea-pig and rat ileum, and could therefore be used as a marker for intrinsic primary afferent neurons. The neurochemical coding of CALB-containing myenteric and submucosal neurons in ileum of unweaned lamb and mature sheep and its co-localisation with various neural markers was studied immunohistochemically. An antiserum against neuronal nuclear protein (NeuN) failed to detect the entire neuronal population; it was expressed only in 48% of neuron-specific enolase (NSE)-immunoreactive (NSE-IR) neurons. Human neuronal protein appeared to occur in the large majority or all neurons. Almost all CALB-IR neurons were: (1) radially multidendritic; (2) eccentric multidendritic; (3) Dogiel type II. CALB-IR occurred in 20-25% of myenteric and 65-75% of submucosal neurons in lamb and mature sheep, with higher values in mature sheep. Nearly all CALB-IR neurons were common choline acetyltransferase (cChAT)-IR, whereas only about 20% of cChAT-IR somata were CALB-IR. In lamb and mature sheep, 90% of MP CALB-IR neurons were peripheral choline acetyltransferase (pChAT)-IR. In lamb SMP, 80±13% of CALB-IR cells were also pChAT-IR, whereas all those in mature SMP were pChAT-IR. Fewer myenteric CALB-IR neurons exhibited tachykinin (TK) in mature sheep (49%) than in lamb (88%). This was also the case for submucosal ganglia (mature sheep, 63%; lamb, 89%). In lamb MP, 77±7% of CALB-IR cells were NeuN-positive. In mature sheep, 73±10% of CALB-IR somata were NeuN-IR, but NeuN failed to stain SMP neurons. In the MP of suckling and mature sheep, Dogiel type II CALB-IR neurons were calcitonin gene-related peptide (CGRP)-IR. In the SMP at both stages, Dogiel type II CALB-IR somata (about 50% of CALB-IR neurons) were also CGRP-IR. Only small proportions of CALB-IR neurons showed immunoreactivity for calretinin or nitric oxide synthase (NOS), although large populations of CALB and NOS neurons occurred in the ganglia. Thus, CALB is a marker of most Dogiel type II neurons in the sheep but is not confined to Dogiel II neurons. CGRP is a more selective marker of Dogiel type II neurons, being only found in this neuron type. [ABSTRACT FROM AUTHOR]

Simple Summary: Nutritional strategies focused on the use of botanicals as modulators of several physiological responses and health promoters of the gastrointestinal tract have attracted interest in animal production. Previous research indicates the positive results of using essential oils (EOs) as natural feed additives for several farmed animals. In the last decades, these nutritional alternatives have been evaluated and reported in fish production in order to increase fish growth and feed utilization and to promote animal welfare. Therefore, the present study was designed to compare the effects of feed EO supplementation in two different forms (natural and composed of active ingredients obtained by synthesis) on the gastric mucosa in European sea bass. EOs decrease oxyntopeptic cells and increase somatostatin and ghrelin enteroendocrine cells. In addition, we showed that Na+K+-ATPase was expressed in oxyntopeptic cells (OPs) in the same way as H+K+-ATPase (typical marker for mammalian parietal cells) and, for this reason, consider Na+K+-ATPase a valid marker for OPs. The current work was designed to assess the effect of feed supplemented with essential oils (EOs) on the histological features in sea bass's gastric mucosa. Fish were fed three diets: control diet (CTR), HERBAL MIX® made with natural EOs (N-EOs), or HERBAL MIX® made with artificial EOs obtained by synthesis (S-EOs) during a 117-day feeding trial. Thereafter, the oxyntopeptic cells (OPs) and the ghrelin (GHR) and somatostatin (SOM) enteroendocrine cells (EECs) in the gastric mucosa were evaluated. The Na+K+-ATPase antibody was used to label OPs, while, for the EECs, anti-SOM and anti-GHR antibody were used. The highest density of OP immunoreactive (IR) area was in the CTR group (0.66 mm2 ± 0.1). The OP-IR area was reduced in the N-EO diet group (0.22 mm2 ± 1; CTR vs. N-EOs, p < 0.005), while in the S-EO diet group (0.39 mm2 ± 1) a trend was observed. We observed an increase of the number of SOM-IR cells in the N-EO diet (15.6 ± 4.2) compared to that in the CTR (11.8 ± 3.7) (N-EOs vs. CTR; p < 0.05), but not in the S-EOs diet. These observations will provide a basis to advance current knowledge on the anatomy and digestive physiology of this species in relation to pro-heath feeds. [ABSTRACT FROM AUTHOR]