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Background and Objectives: Some blood operators routinely screen blood donations for high-titre (HT) anti-A/B to reduce the risk of a haemolytic transfusion reaction due to out-of-group plasma-rich components. We assessed donor factors associated with an increased likelihood of screening positive and compared routine data between England and Australia., Materials and Methods: Data were assessed from HT screening during 2018-2020 in Australia and 2018-2021 in England, totalling nearly 6 million blood donations. Screening was performed using a Beckman Coulter PK7300 analyser with a micro-titre plate saline direct agglutination test in both countries, although different reagent red cells were chosen. HT-positive was defined as testing positive at a titre of 128 or above., Results: The likelihood of a donor testing HT-positive was greater for females than males, declined with age and was dependent on the ABO group. However, the proportion of donors testing HT-positive was consistently higher in Australia than in England: overall, 14% of group O donations and 5% of group A donations in England tested HT-positive, compared with 51% and 22%, respectively in Australia. English data also showed that donors from Black, Asian or mixed ethnic backgrounds were more likely to test HT-positive than White donors., Conclusion: These data demonstrate that donor sex, age, ABO group and ethnicity affect the likelihood of testing HT-positive. Differences in testing methods likely had a significant impact on the proportion of donors testing as HT-positive or -negative rather than any differences in donor populations., (© 2024 The Author(s). Vox Sanguinis published by John Wiley & Sons Ltd on behalf of International Society of Blood Transfusion.)

Background: We report an obstetric case involving an RhD-positive woman who had developed a red blood cell (RBC) antibody that was not detected until after delivery of a newborn, who presented with a positive direct antiglobulin test result. Immunohematology studies suggested that the maternal antibody was directed against a low-prevalence antigen on the paternal and newborn RBCs., Results: Comprehensive blood group profiling by targeted exome sequencing revealed a novel nonsynonymous single nucleotide variant (SNV) RHCE c.486C>G (GenBank MZ326705) on the RHCE*Ce allele, for both the father and newborn. A subsequent genomic-based study to profile blood groups in an Indigenous Australian population revealed the same SNV in 2 of 247 individuals. Serology testing showed that the maternal antibody reacted specifically with RBCs from these two individuals., Discussion: The maternal antibody was directed against a novel antigen in the Rh blood group system arising from an RHCE c.486C>G variant on the RHCE*Ce allele linked to RHD*01. The variant predicts a p.Asn162Lys change on the RhCE protein and has been registered as the 56th antigen in the Rh system, ISBT RH 004063., Conclusion: This antibody was of clinical significance, resulting in a mild to moderate hemolytic disease of the fetus and newborn (HDFN). In the past, the cause of such HDFN cases may have remained unresolved. Genomic sequencing combined with population studies now assists in resolving such cases. Further population studies have potential to inform the need to design population-specific red cell antibody typing panels for antibody screening in the Australian population., (© 2022 AABB.)

Background: Australian Red Cross Lifeblood (Lifeblood) performs human erythrocyte antigen (HEA) genotyping for a subset of repeat whole-blood donors through preferential selection which aims to maximise variation of results and possibility of identifying donors lacking high frequency red cell antigens., Materials and Methods: The HEA Molecular Bead chip™ assay is used by Lifeblood for donor genotyping. A review of all donor HEA genotype data from March 2019 to May 2022 (3 years) was conducted., Results: HEA genotyping was performed for 20,185donors. Due to selective genotyping of donors, a higher frequency of R1R1 predicted phenotype was identified. However, frequencies of other red cell phenotypes were relatively similar to previous reported in the Australian population. A small number of donors with rare red cell phenotypes was identified., Conclusion: Genotyping of blood donors provides an available pool of extended matched red blood cell products for matching to recipients. Additionally genotyping can improve the identification of donors with rare phenotypes. Whilst limitations exist, genotyping may reduce the need for labour intensive serotyping, improve blood inventory management, and may be useful in donor recruitment and retention., (© 2024 Australian Red Cross Lifeblood. Transfusion Medicine published by John Wiley & Sons Ltd on behalf of British Blood Transfusion Society.)

Red blood cell (RBC) transfusion can cause some patients to form antibodies to RBC antigens when RBC phenotypes do not match that of the blood donor. Transfusion practitioners can order phenotyped RBC units for patients with known RBC antibodies or those who are at risk of forming them. However, with increasing demand for phenotyped RBC units, contemporary data on antigen prevalence is required to manage the changing supply. A total of 490,491 blood donors, including 103,798 (21.2%) first-time blood donors, from 2019 were analysed for the prevalence of selected clinically relevant blood group antigens. Prevalence of the phenotype R 1 R 1 (D+ C+ E- c- e+) increased from the previous estimate of 17.3% to 24.0% in first-time blood donors. The prevalence of R 1 r (D+ C+ E- c+ e+) decreased from 35.3% to 30.8%. R 1 R 1 was more common in blood donors born in Asia or the Middle East. The prevalence of Fy(a-b-) in donors where Fy antigens were tested was 0.2%. Of these, 71.8% stated their region of birth as Africa. The prevalence of Jk(a-b-) is 0.01% in donors where the Jk antigens were tested with region of birth stated as either Oceania or Asia. The increasing prevalence of the c-negative phenotype in R 1 R 1 individuals is associated with the changing demographics of the Australian community. For R 1 R 1 individuals with childbearing potential, the transfusion of RhD negative blood, which is usually c-positive, may increase the possibility of haemolytic disease of the fetus and newborn during pregnancy. Continued diversification of the Australian blood donor panel will support having the appropriate phenotyped RBC units available., (Copyright © 2023 Royal College of Pathologists of Australasia. All rights reserved.)

Background and Objectives: A newborn presented with jaundice in Thailand. The cord red cells tested positive by direct antiglobulin test (DAT) for an unknown maternal red cell antibody. Initial blood group sequencing suggested that the infant carried a novel variant RHAG c.140T>C, responsible for a low-prevalence antigen in the RHAG blood group system (ISBT 030). We report here on testing of samples from the infant's parents and older sibling to define a new antigen in the RHAG system., Materials and Methods: Massive parallel sequencing (MPS) using a custom-designed panel was performed on all four family members. Extended serological testing was also performed to determine whether family members with the same variant as the infant showed reactivity with the antibody in the maternal plasma., Results: We identified a novel single nucleotide variant (SNV) (RHAG c.140T>C, p.[Phe47Ser]) in samples from three of the four family members tested (the infant, the older sibling and the father). The variant was not detected in the mother's sample. Maternal plasma showed positive agglutination with all family members tested; however, when tested with routine panel cells, no reactivity was observed., Conclusion: This case study showed that the presence of the novel variant (RHAG c.140T>C), encoding a p.(Phe47Ser) change in the RhAG glycoprotein, was the apparent cause of incompatibility between maternal plasma and that of red cells from the proband, father and older sibling of the proband. We propose this variant to be a new low-prevalence antigen in the RHAG blood group system., (© 2023 The Authors. Vox Sanguinis published by John Wiley & Sons Ltd on behalf of International Society of Blood Transfusion.)

Vagal preganglionic neurons innervate myenteric ganglia. These autonomic efferents are distributed so densely within the ganglia that it has been impractical to track individual vagal axons through the myenteric plexus with tracer labeling. To evaluate whether vagal efferent axons evidence selectivity, particularly for nitrergic or non-nitrergic myenteric neurons within the plexus, we limited the numbers and volumes of brainstem dextran biotin tracer injections per animal. Reduced labeling and the use of immunohistochemistry generated cases in which some individual axons could be distinguished and traced in three dimensions (Neurolucida) within and among successive (up to 46) myenteric ganglia. In the myenteric plexus of all stomach regions, the majority (∼86%) of vagal efferents were organized into two distinct subtypes. One subtype (∼24% of dextran-labeled efferents, designated "primarily nitrergic") selectively contacted and linked-both within and between ganglia-nitric oxide synthase positive (nNOS+) neurons into presumptive motor modules. A second subtype (∼62% of efferents, designated "primarily non-nitrergic") appeared to selectively contact and link-both within and between ganglia-non-nitrergic enteric neurons into a second type of effector ensemble. A third candidate type (∼14% of labeled preganglionics), appeared to lack "nitrergic selectivity" and to contact both nNOS+ and nNOS- enteric neurons. In addition to the quantitative assessment of the efferent axons in stomach, qualitative observations of the proximal duodenum indicated similar selective vagal efferent projections, in proportions comparable with those evaluated in the stomach. Limited injections of tracer, three-dimensional (3-D) tracing of individual axons, and histochemistry of myenteric neurons might distinguish additional efferent phenotypes. NEW & NOTEWORTHY The present study highlights the following: 1 ) one type of vagal efferent axon selectively innervates nitrergic upper gastrointestinal myenteric neurons; 2 ) a second type of vagal efferent selectively innervates non-nitrergic gastrointestinal myenteric neurons; and 3 ) the two types of vagal efferents might modulate peristalsis reciprocally and cooperatively.

Nociceptive afferents innervate the stomach and send signals centrally to the brain and locally to stomach tissues. Nociceptive afferents can be detected with a variety of different markers. In particular, substance P (SP) is a neuropeptide and is one of the most commonly used markers for nociceptive nerves in the somatic and visceral organs. However, the topographical distribution and morphological structure of SP-immunoreactive (SP-IR) axons and terminals in the whole stomach have not yet been fully determined. In this study, we labeled SP-IR axons and terminals in flat mounts of the ventral and dorsal halves of the stomach of mice. Flat-mount stomachs, including the longitudinal and circular muscular layers and the myenteric ganglionic plexus, were processed with SP primary antibody followed by fluorescent secondary antibody and then scanned using confocal microscopy. We found that (1) SP-IR axons and terminals formed an extensive network of fibers in the muscular layers and within the ganglia of the myenteric plexus of the whole stomach. (2) Many axons that ran in parallel with the long axes of the longitudinal and circular muscles were also immunoreactive for the vesicular acetylcholine transporter (VAChT). (3) SP-IR axons formed very dense terminal varicosities encircling individual neurons in the myenteric plexus; many of these were VAChT immunoreactive. (4) The regional density of SP-IR axons and terminals in the muscle and myenteric plexus varied in the following order from high to low: antrum-pylorus, corpus, fundus, and cardia. (5) In only the longitudinal and circular muscles, the regional density of SP-IR axon innervation from high to low were: antrum-pylorus, corpus, cardia, and fundus. (6) The innervation patterns of SP-IR axons and terminals in the ventral and dorsal stomach were comparable. Collectively, our data provide for the first time a map of the distribution and morphology of SP-IR axons and terminals in the whole stomach with single-cell/axon/synapse resolution. This work will establish an anatomical foundation for functional mapping of the SP-IR axon innervation of the stomach and its pathological remodeling in gastrointestinal diseases., (© 2022 Wiley Periodicals LLC.)

In this study, a new air-assisted flare tip was designed, built, and tested under different operating conditions. Lacking sufficient energy to mix with air, low waste gas flow rates will lead to incomplete combustion of these gases. This increases pollutant emissions and soot formation which leads to a decline in flare performance. This flare tip design enhances the waste gas mixing energy through implementation of an air jet in a crossflow orientation. This is done by adjusting the exit area of the waste gas exit by injecting a radial jet of air from an inclined slot jet located around the flare tip. This flare tip design also provides protection for the flare tip from high flame temperatures that can damage through convective cooling. Several tests were conducted to assess the new flare tip design with varying waste gas flow rates of 5, 10, 25, and 120 standard liters per minute (SLPM). These tests also considered varying assistant air flow rates. In addition, test results showed high combustion efficiency of the flaring process and significant soot formation suppression. The new flare tip design yielded better flame behavior with respect to the flare tip, caused by the flame stability that prevented the flame from attaching to the flare tip., Competing Interests: The authors declare no competing financial interest., (© 2022 The Authors. Published by American Chemical Society.)

In the last decade, Australia has experienced an overall decline in red cell demand, but there has been an increased need for phenotyped matched red cells. Lifeblood and mathematicians from Queensland universities have developed a probabilistic model to determine the percentage of the donor panel that would need extended antigen typing to meet this increasing demand, and an estimated timeline to achieve the optimum required phenotyped (genotyped) panel. Mathematical modelling, based on Multinomial distributions, was used to provide guidance on the percentage of typed donor panel needed, based on recent historical blood request data and the current donor panel size. Only antigen combinations determined to be uncommon, but not rare, were considered. Simulations were run to attain at least 95% success percentage. Modelling predicted a target of 38% of the donor panel, or 205,000 donors, would need to be genotyped to meet the current demand. If 5% of weekly returning donors were genotyped, this target would be reached within 12 years. For phenotyping, 35% or 188,000 donors would need to be phenotyped to meet Lifeblood's demand. With the current level of testing, this would take eight years but could be performed within three years if testing was increased to 9% of weekly returning donors. An additional 26,140 returning donors need to be phenotyped annually to maintain this panel. This mathematical model will inform business decisions and assist Lifeblood in determining the level of investment required to meet the desired timeline to achieve the optimum donor panel size., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2022 Best et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Background and Objectives: Red cell antigen genotyping is commonly performed on patients requiring chronic transfusion support, such as sickle cell disease and thalassaemia. The Immucor HEA BeadChip™ test, in addition to assessing red cell antigen expression, can also detect the haemoglobin S (HbS) mutation. Our aim was to compare HbS results using HEA BeadChip™ performed at the Australian Red Cross Lifeblood with conventional haemoglobin studies., Materials and Methods: Patients with thalassaemia and sickle cell trait (SCT) or disease (SCD) referred for red cell genotyping between 2017 and 2019 were assessed. The HbS result obtained from HEA BeadChip™ was compared with that obtained from high-performance liquid chromatography (HPLC) performed by the referring pathology provider., Results: One-hundred and nineteen cases had comparable HPLC and HEA BeadChip™ results. On HEA BeadChip™ testing, 40 cases showed a negative HbS result, 31 cases showed HbS+ and 47 cases showed HbS++. There was one case with 'low signal' result. Of the negative HbS cases, there was none with SCT. The HbS+ group comprised a mixture of SCT and SCD due to compound heterozygosity for HbS and β-thalassaemia mutations. The HbS++ group comprised predominantly SCD due to homozygosity for HbS., Conclusion: HEA BeadChip™ is an accurate screening test for the detection of HbS. There were no false positives or false negatives. The identification of donors with the HbS mutation through the targeted genotyping programme would enable early intervention, improved donor management and reduced wastage., (© 2022 International Society of Blood Transfusion.)

Background and Objectives: Australian Red Cross Lifeblood (Lifeblood) performs red blood cell (RBC) antibody screening on every whole blood donation. An alternate strategy has been proposed whereby an antibody screen is performed on the first donation and only repeated following pregnancy, transfusion or a significant break between donations (>2 years). We assess the blood safety risks associated with removing antibody screening for every whole blood donation., Materials and Methods: A retrospective desktop analysis included all whole blood donations collected by Lifeblood between 01 May 2018 and 30 April 2019 to quantify the antibodies that would have been undetected with the alternate strategy. The strategy was further assessed using the Alliance of Blood Operators Risk-Based Decision-Making framework., Results: One hundred and seventy-one routine donors had antibodies for the first time, but reported no sensitizing event since their last donation. Forty-seven of these had antibodies of a clinically significant specificity and titre that have the potential to cause a haemolytic transfusion reaction (HTR). The calculated risk of undetected antibodies being transfused to an incompatible recipient is 1 in 82,200., Conclusion: The estimated risk of HTRs with the alternate strategy results in an increased risk. While the alternate strategy is identified as the most cost-effective option within the Australian setting, this additional residual risk was not deemed to be acceptable. Blood services would need to determine whether the increase in residual risk stemming from implementation of such a strategy is tolerable., (© 2021 International Society of Blood Transfusion.)

Maternal alloimmunisation against red blood cell antigens can cause haemolytic disease of the fetus and newborn (HDFN). Although most frequently caused by anti-D, since the implementation of rhesus D (RhD) immunoglobulin prophylaxis, other alloantibodies have become more prevalent in HDFN. Recent advances in non-invasive prenatal testing (NIPT) have allowed early prediction of HDFN risk in alloimmunised pregnancies and allow clinicians to focus health resources on those pregnancies that require intervention. This article aims to provide updates on the current status of NIPT in Australia as both a diagnostic and screening tool in pregnancy., (© 2021 The Royal Australian and New Zealand College of Obstetricians and Gynaecologists.)

Axonal characterizations of connectomes in healthy and disease phenotypes are surprisingly incomplete and biased because unmyelinated axons, the most prevalent type of fibers in the nervous system, have largely been ignored as their quantitative assessment quickly becomes unmanageable as the number of axons increases. Herein, we introduce the first prototype of a high-throughput processing pipeline for automated segmentation of unmyelinated fibers. Our team has used transmission electron microscopy images of vagus and pelvic nerves in rats. All unmyelinated axons in these images are individually annotated and used as labeled data to train and validate a deep instance segmentation network. We investigate the effect of different training strategies on the overall segmentation accuracy of the network. We extensively validate the segmentation algorithm as a stand-alone segmentation tool as well as in an expert-in-the-loop hybrid segmentation setting with preliminary, albeit remarkably encouraging results. Our algorithm achieves an instance-level [Formula: see text] score of between 0.7 and 0.9 on various test images in the stand-alone mode and reduces expert annotation labor by 80% in the hybrid setting. We hope that this new high-throughput segmentation pipeline will enable quick and accurate characterization of unmyelinated fibers at scale and become instrumental in significantly advancing our understanding of connectomes in both the peripheral and the central nervous systems., (© 2022. The Author(s).)

Background: Australian Red Cross Lifeblood has seen a 50 % increase in demand for phenotyped red blood cell (RBC) units between 2016-2018 and a 30 % increase in demand in 2018 to perform molecular RBC typing on patient samples. Lifeblood conducted a survey to understand transfusion laboratory practices for requesting patient phenotyping and/or molecular RBC typing and for selecting phenotyped RBC units in various patient groups., Study Design and Methods: An electronic Qualtrics survey form was sent to 296 transfusion laboratories with questions designed to understand the practice of selecting phenotyped RBC units and reasons for requesting extended serology or molecular RBC typing., Results: 49 (16.6 %) transfusion laboratories provided data. Reasons to request extended phenotyping and/or molecular RBC typing for patients included; chronic transfusion (n = 31 laboratories), sickle cell disease (n = 25), Thalassemia (n = 23), requirement for anti-CD38 or other MAB therapy (n = 23) or Myelodysplasia (n = 22). Forty-seven transfusion laboratories provided responses with reasons for requesting molecular RBC typing which included: predicting phenotype in patients with multiple antibodies (n = 31), prior to administering anti-CD38 or other MAB therapies (n = 29), for pregnancy related transfusions (n = 28) or for confirming the phenotype of recently transfused patients (n = 18)., Conclusion: Transfusion laboratory practices indicated that phenotyped RBC units were selected for patients requiring chronic transfusion support and/or undergoing MAB therapy. Requests for molecular RBC typing occurred for more complex patient requirements where serological investigations were not suitable or possible due to reagent restrictions., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Background: Vagus nerve stimulation (VNS) is an emerging electroceutical therapy for remedying gastric disorders that are poorly managed by pharmacological treatments and/or dietary changes. Such therapy seems promising as the vagovagal neurocircuitry modulates the enteric nervous system to influence gastric functions., Methods: Here, the modulatory effects of left cervical VNS on gastric emptying in rats were quantified using a (i) feeding protocol in which the animal voluntarily consumed a postfast, gadolinium-labeled meal and (ii) a non-invasive imaging method to measure antral motility, pyloric activity and gastric emptying based on contrast-enhanced magnetic resonance imaging (MRI) and computer-assisted image processing pipelines., Key Results: Vagus nerve stimulation significantly accelerated gastric emptying (sham vs VNS: 29.1% ± 1.5% vs 40.7% ± 3.9% of meal emptied per 4 hours), caused a greater relaxation of the pyloric sphincter (sham vs VNS: 1.5 ± 0.1 vs 2.6 ± 0.4 mm 2 cross-sectional area of lumen), and increased antral contraction amplitude (sham vs VNS: 23.3% ± 3.0% vs 32.5% ± 3.0% occlusion), peristaltic velocity (sham vs VNS: 0.50 ± 0.02 vs 0.67 ± 0.03 mm s -1 ), but not its contraction frequency (sham vs VNS: 6.1 ± 0.2 vs 6.4 ± 0.2 contractions per minute, P = .22). The degree to which VNS relaxed the pylorus was positively correlated with gastric emptying rate (r = .5887, P < .001)., Conclusions & Inferences: The MRI protocol employed in this study is expected to enable advanced preclinical studies to understand stomach pathophysiology and its therapeutics. Results from this study suggest an electroceutical treatment approach for gastric emptying disorders using cervical VNS to control the degree of pyloric sphincter relaxation., (© 2018 John Wiley & Sons Ltd.)

Background: We previously demonstrated that targeted exome sequencing accurately defined blood group genotypes for reference panel samples characterized by serology and single-nucleotide polymorphism (SNP) genotyping. Here we investigate the application of this approach to resolve problematic serology and SNP-typing cases., Study Design and Methods: The TruSight One sequencing panel and MiSeq platform was used for sequencing. CLC Genomics Workbench software was used for data analysis of the blood group genes implicated in the serology and SNP-typing problem. Sequence variants were compared to public databases listing blood group alleles. The effect of predicted amino acid changes on protein function for novel alleles was assessed using SIFT and PolyPhen-2., Results: Among 29 unresolved samples, sequencing defined SNPs in blood group genes consistent with serologic observation: 22 samples exhibited SNPs associated with varied but known blood group alleles and one sample exhibited a chimeric RH genotype. Three samples showed novel variants in the CROM, LAN, and RH systems, respectively, predicting respective amino acid changes with possible deleterious impact. Two samples harbored rare variants in the RH and FY systems, respectively, not previously associated with a blood group allele or phenotype. A final sample comprised a rare variant within the KLF1 transcription factor gene that may modulate DNA-binding activity., Conclusion: Targeted exome sequencing resolved complex serology problems and defined both novel blood group alleles (CD55:c.203G>A, ABCB6:c.1118&#95;1124delCGGATCG, ABCB6:c.1656-1G>A, and RHD:c.452G>A) and rare variants on blood group alleles associated with altered phenotypes. This study illustrates the utility of exome sequencing, in conjunction with serology, as an alternative approach to resolve complex cases., (© 2017 AABB.)

Background: Blood group single nucleotide polymorphism genotyping probes for a limited range of polymorphisms. This study investigated whether massively parallel sequencing (also known as next-generation sequencing), with a targeted exome strategy, provides an extended blood group genotype and the extent to which massively parallel sequencing correctly genotypes in homologous gene systems, such as RH and MNS., Study Design and Methods: Donor samples (n = 28) that were extensively phenotyped and genotyped using single nucleotide polymorphism typing, were analyzed using the TruSight One Sequencing Panel and MiSeq platform. Genes for 28 protein-based blood group systems, GATA1, and KLF1 were analyzed. Copy number variation analysis was used to characterize complex structural variants in the GYPC and RH systems., Results: The average sequencing depth per target region was 66.2 ± 39.8. Each sample harbored on average 43 ± 9 variants, of which 10 ± 3 were used for genotyping. For the 28 samples, massively parallel sequencing variant sequences correctly matched expected sequences based on single nucleotide polymorphism genotyping data. Copy number variation analysis defined the Rh C/c alleles and complex RHD hybrids. Hybrid RHD*D-CE-D variants were correctly identified, but copy number variation analysis did not confidently distinguish between D and CE exon deletion versus rearrangement., Conclusion: The targeted exome sequencing strategy employed extended the range of blood group genotypes detected compared with single nucleotide polymorphism typing. This single-test format included detection of complex MNS hybrid cases and, with copy number variation analysis, defined RH hybrid genes along with the RHCE*C allele hitherto difficult to resolve by variant detection. The approach is economical compared with whole-genome sequencing and is suitable for a red blood cell reference laboratory setting., (© 2017 AABB.)

Background: This experiment assessed normative expression patterns of alpha-synuclein (SYNC), including ganglionic remodeling and development of SYNC pathologies, in the submucosal plexus (SMP) of the colon during healthy aging. The observations address age-associated changes in bowel function and are relevant to evaluations of SMP-containing colonic biopsies for SYNC or synucleinopathies associated with aging and peripheral neurodegenerative diseases., Methods: Colonic submucosal whole mounts from groups of virgin male Fischer 344 rats (n ≥ 8 per group) at 4, 8, 16, and 24 months of age were processed immunohistochemically for SYNC and the pan-neuronal marker HuC/D. In addition, macrophages immunoreactive for MHCII were examined. Stereological protocols were used to generate unbiased estimates of neuron density, neurons per ganglion, neurons per ganglionic area, and neuron size., Key Results: The protein SYNC was expressed in a subpopulation of SMP neurons, in both nucleus and cytoplasm. The general age-associated pattern across different cell counts was an increase in the number of SYNC+ neurons between 4 and 8 months of age, with progressively decreasing numbers of both SYNC+ and SYNC- neurons over the remaining lifespan. The soma size of SYNC+ neurons increased progressively with age. Aggregated SYNC occurred in the aging SMP, and macrophages with alternatively activated profiles were located adjacent to pathological SYNC deposits, consistent with ongoing phagocytosis., Conclusions & Inferences: Changes in SYNC expression with age, including a baseline of accumulating synucleinopathies in the healthy aging SMP, need to be considered when interpreting either functional disturbances or biopsies of the aging colon., (© 2013 John Wiley & Sons Ltd.)

Background: The gastric sling muscle has not been investigated for possible sensory innervation, in spite of the key roles the structure plays in lower esophageal sphincter (LES) function and gastric physiology. Thus, the present experiment used tracing techniques to label vagal afferents and survey their projections in the lesser curvature., Methods: Sprague-Dawley rats received injections of dextran biotin into the nodose ganglia. Fourteen days postinjection, animals were euthanized and their stomachs were processed to visualize the vagal afferent innervation. In different cases, neurons, muscle cells, or interstitial cells of Cajal (ICC) were counterstained., Key Results: The sling muscle is innervated throughout its length by vagal afferent intramuscular arrays (IMAs) associated with ICC. In addition, the distal antral attachment site of the sling muscle is innervated by a novel vagal afferent terminal specialization, an antral web ending. The muscle wall of the distal antrum is also innervated by conventional IMAs and intraganglionic laminar endings, the two types of mechanoreceptors found throughout stomach smooth muscle., Conclusions & Inferences: The innervation of sling muscle by IMAs, putative stretch receptors, suggests that sling sensory feedback may generate vago-vagal or other reflexes with vagal afferent limbs. The restricted distribution of afferent web endings near the antral attachments of sling fibers suggests the possibility of specialized mechanoreceptor functions linking antral and pyloric activity to the operation of the LES. Dysfunctional sling afferents could generate LES motor disturbances, or normative compensatory sensory feedback from the muscle could compromise therapies targeting only effectors., (© 2012 Blackwell Publishing Ltd.)

Intramuscular arrays (IMAs), vagal mechanoreceptors that innervate gastrointestinal smooth muscle, have not been completely described structurally or functionally. To delineate more fully the architecture of IMAs and to consider the structure-function implications of the observations, the present experiment examined the organization of the IMA terminal arbors and the accessory tissue elements of those arbors. IMA terminal fields, labeled by injection of biotinylated dextran into the nodose ganglia, were examined in whole mounts of rat gastric smooth muscle double-labeled with immunohistochemistry for interstitial cells of Cajal (ICCs; c-Kit) and/or inputs of different neuronal efferent transmitter (markers: tyrosine hydroxylase (TH), vesicular acetylcholine transporter (VAChT), and nitric oxide synthase (NOS)) or afferent neuropeptidergic (calcitonin gene-related peptide (CGRP)) phenotypes. IMAs make extensive varicose and lamellar contacts with ICCs. In addition, axons of the multiple efferent and afferent phenotypes examined converge and articulate with IMA terminal arbors innervating ICCs. This architecture is consistent with the hypothesis that IMAs, or the multiply innervated IMA-ICC complexes they form, can function as stretch receptors. The tissue organization is also consonant with the proposal that those units can operate as functional analogues of muscle spindle organs. For electrophysiological assessments of IMA functions, experiments will need protocols that preserve both the complex architecture and the dynamic operations of IMA-ICC complexes., (Copyright © 2011 IBRO. Published by Elsevier Ltd. All rights reserved.)

Background and Aims: Several types of gastric surgeries have been associated with early satiety, dyspepsia and food intolerances. We aimed to examine alterations in gastric vagal afferents following gastric surgery-fundus ligation., Methods: Six week old, male Sprague-Dawley rats underwent chronic ligation (CL) of the fundus. Sham rats underwent abdominal surgery, but without ligation. Another group of rats underwent acute ligation (AL) of the fundus immediately prior to experiments. CL and sham rats were allowed to grow to age 3-4 months. Food intake and weights were recorded post-operatively. Gastric compliance and gastric wall thickness was measured at baseline and during gastric distension (GD). Extracellular recordings were made to examine response characteristics of vagal afferent fibers to GD and to map the stomach receptive field (RF). The morphological structures of afferent terminals in the stomach were examined with retrograde tracings from the nodose ganglion., Results: The CL group consumed significantly less food and weighed less than sham control. The mean compliance of the CL group was significantly less than control, but higher than the AL group. The spontaneous firing and responses to GD of afferent fibers from the CL rats were significantly higher than AL rats. There was a marked expansion of the gastric RF in the CL rats with significant reorganization and regeneration of intramuscular array (IMA) terminals. There was no difference in total wall or muscle thickness among the groups., Conclusion: CL results in aberrant remodeling of IMAs with expansion of the gastric RF and alters the mechanotransduction properties of vagal afferent fibers. These changes could contribute to altered sensitivity following gastric surgery.

The protein alpha-synuclein is implicated in the development of Parkinson's disease. The molecule forms Lewy body aggregates that are hallmarks of the disease, has been associated with the spread of neuropathology from the peripheral to the CNS, and appears to be involved with the autonomic disorders responsible for the gastrointestinal (GI) symptoms of individuals afflicted with Parkinson's. To characterize the normative expression of alpha-synuclein in the innervation of the GI tract, we examined both the postganglionic neurons and the preganglionic projections by which the disease is postulated to retrogradely invade the CNS. Specifically, in Fischer 344 and Sprague-Dawley rats, immunohistochemistry in conjunction with injections of the tracer Dextran-Texas Red was used to determine, respectively, the expression of alpha-synuclein in the myenteric plexus and in the vagal terminals. Alpha-synuclein is expressed in a subpopulation of myenteric neurons, with the proportion of positive somata increasing from the stomach (approximately 3%) through duodenum (proximal, approximately 6%; distal, approximately 13%) to jejunum (approximately 22%). Alpha-synuclein is co-expressed with the nitrergic enzyme nitric oxide synthase (NOS) or the cholinergic markers calbindin and calretinin in regionally specific patterns: approximately 90% of forestomach neurons positive for alpha-synuclein express NOS, whereas approximately 92% of corpus-antrum neurons positive for alpha-synuclein express cholinergic markers. Vagal afferent endings in the myenteric plexus and the GI smooth muscle do not express alpha-synuclein, whereas, virtually all vagal preganglionic projections to the gut express alpha-synuclein, both in axons and in terminal varicosities in apposition with myenteric neurons. Vagotomy eliminates most, but not all, alpha-synuclein-positive neurites in the plexus. Some vagal preganglionic efferents expressing alpha-synuclein form varicose terminal rings around myenteric plexus neurons that are also positive for the protein, thus providing a candidate alpha-synuclein-expressing pathway for the retrograde transport of putative Parkinson's pathogens or toxins from the ENS to the CNS.

To assess whether afferent vagal intramuscular arrays (IMAs), putative gastrointestinal mechanoreceptors, form contacts with interstitial cells of Cajal of the intramuscular type (ICC-IM) and to describe any such contacts, electron microscopic analyses were performed on the external muscle layers of the fundus containing dextran-labelled diaminobenzidin (DAB)-stained IMAs. Special staining and embedding techniques were developed to preserve ultrastructural features. Within the muscle layers, IMA varicosities were observed in nerve bundles traversing major septa without contact with ICC-IM, contacting unlabelled neurites and glial cells. IMA varicosities were encountered in minor septa in contact with ICC-IM which were not necessarily in close contact with muscle cells. In addition, IMA varicosities were observed within muscle bundles in close contact with ICC-IM which were in gap junction contact with muscle cells. IMAs formed varicosities containing predominantly small agranular vesicles, occasionally large granular vesicles and prejunctional thickenings in apposition to ICC-IM processes, indicating communication between ICC and IMA via synapse-like contacts. Taken together, these different morphological features are consistent with a hypothesized mechanoreceptor role for IMA-ICC complexes. Intraganglionic laminar ending varicosities contacted neuronal somata and dendrites in the myenteric plexus of the fundus, but no contacts with ICC associated with Auerbach's plexus were encountered.

The relative potencies of cholecystokinin (CCK)-8 and CCK-33 for decreasing meal size depend on the route of administration. Inhibitory potencies are equal after intraperitoneal administration, but CCK-33 is significantly more potent after intraportal administration. This suggests that CCK-33 is a more effective stimulant of hepatic afferent vagal nerves than is CCK-8. To investigate this possibility, we administered both peptides intraperitoneally in rats with abdominal vagotomies that spared only the hepatic proper vagal nerves (H) and in rats with abdominal vagotomies that spared the common hepatic branch that contains the fibers of the hepatic proper and gastroduodenal nerves (HGD). The vagal afferent innervation in H and HGD rats was verified with a wheat germ agglutinin-horseradish tracer strategy. Intraperitoneal administration of CCK-33 decreased 30-min intake of 10% sucrose in H rats as much as in sham rats, but CCK-8 decreased intake significantly less in H rats than in sham rats. The larger inhibitory effect of CCK-33 than of CCK-8 in H rats is consistent with the hypothesis that CCK-33 is a more effective stimulant of the hepatic proper vagal afferent nerves than CCK-8. In contrast to the results in H rats, the inhibitory potencies of both peptides were significantly and equivalently reduced in HGD rats compared with sham rats. This suggests that there is an inhibitory interaction between the stimulation of the gastroduodenal and hepatic proper afferent fibers by CCK-33.

Intraganglionic laminar endings (IGLEs) and intramuscular arrays (IMAs) are the two putative mechanoreceptors that the vagus nerve supplies to gastrointestinal smooth muscle. To examine whether neurotrophin-4 (NT-4)-deficient mice, which have only 45% of the normal number of nodose ganglion neurons, exhibit selective losses of these endings and potentially provide a model for assessing their functional roles, we inventoried IGLEs and IMAs in the gut wall. Vagal afferents were labeled by nodose ganglion injections of wheat germ agglutinin-horseradish peroxidase, and a standardized sampling protocol was used to map the terminals in the stomach, duodenum, and ileum. NT-4 mutants had a substantial organ-specific reduction of IGLEs; whereas the morphologies and densities of both IGLEs and IMAs in the stomach were similar to wild-type patterns, IGLEs were largely absent in the small intestine (90 and 81% losses in duodenum and ileum, respectively). Meal pattern analyses revealed that NT-4 mutants had increased meal durations with solid food and increased meal sizes with liquid food. However, daily total food intake and body weight remained normal because of compensatory changes in other meal parameters. These findings indicate that NT-4 knock-out mice have a selective vagal afferent loss and suggest that intestinal IGLEs (1) may participate in short-term satiety, probably by conveying feedback about intestinal distension or transit to the brain, (2) are not essential for long-term control of feeding and body weight, and (3) play different roles in regulation of solid and liquid diet intake.

The vagal innervation of the proximal gastrointestinal (GI) tract is lateralized. To determine whether this pattern is specified as early as the perinatal period, neonatal rat pups were given unilateral cervical vagotomies. Separate groups received (1) transections below the left nodose ganglion, (2) left cervical resections that included removal of the nodose ganglion, or (3) sham surgeries. At 4 months of age, each animal's vagal afferent projections from the unoperated side were mapped by injecting the nodose with WGA-HRP, preparing the stomach as wholemounts, and processing the tissue with tetramethyl benzidine. The two types of vagal afferent endings in GI smooth muscle, namely intraganglionic laminar endings and intramuscular arrays, were surveyed separately, and their regional distributions were mapped. Changes in the nucleus of the solitary tract (NST) and dorsal motor nucleus of the vagus (DMNX) were assessed with cell counts and area measurements. Neonatal loss of the vagus innervating one side of the GI tract, with or without ganglionectomy, did not cause the unoperated vagus to sprout to the denervated side. In addition, removal of the projections to the one side of the target organ did not produce a reorganization of the projection maps of the unoperated vagus within its normal or ipsilateral wall of the GI tract. Although the regional patterns of the unoperated ipsilateral vagus were not affected, the packing densities of both types of afferents supplied by this trunk were moderately reduced. The DMNX of the vagotomized side displayed extensive (approximately 83%) neuronal loss; the DMNX on the unoperated side as well as the NST on both sides exhibited limited (approximately 20--25%) losses. The lack of a peripheral projection field reorganization -- except for a moderate down-regulation -- after complete unilateral denervation suggests that both the laterality and the afferent terminal phenotypes (or target tissues) of the vagus in the proximal GI tract are specified by postnatal day one in the rat. The present results, taken together with other observations, also suggest that three different combinations of signals orchestrate the commitments of vagal afferents respectively to (1) the side of the organ, (2) the region within the organ wall, and (3) the accessory and innervated tissues that complex with the fully differentiated ending.

Intramuscular arrays are one of two major classes of vagal afferent mechanoreceptors that innervate the smooth muscle wall of the proximal gastrointestinal tract. They consist of rectilinear telodendria that distribute in the muscle sheets, parallel to the long axes of muscle fibers. Intramuscular arrays appear to make direct contact with the muscle fibers, but they also course on, and form appositions with, intramuscular interstitial cells of Cajal. These complexes formed by intramuscular arrays and intramuscular interstitial cells of Cajal suggest that intramuscular arrays might require either structural or trophic support of the interstitial cells of Cajal for normal differentiation and/or maintenance. To evaluate this hypothesis, we have examined the morphology and distribution of vagal afferent endings in the c-Kit mutant mouse that lacks intramuscular interstitial cells of Cajal. Vagal afferents were labeled by nodose ganglion injection of either wheat germ agglutinin-horseradish peroxidase conjugate or a tagged dextran, and the labeled afferent terminals in the stomach were mapped using a standardized quantitative sampling scheme. Intramuscular arrays were dramatically reduced (in circular muscle by 63%; in longitudinal muscle by 78%) in the c-Kit mutant mice relative to their wild-type littermates. Additionally, a substantial number of the surviving axons and terminals in the mutant stomachs were morphologically aberrant. Moreover, the loss of intramuscular arrays in mutants appeared to be selective: the structure, distribution and density of intraganglionic laminar endings, i.e., the other vagal mechanoreceptors in smooth muscle, were not significantly altered. Finally, the conspicuous decrease in intramuscular array density in mutants was associated with a non-significant trend toward loss of nodose ganglion neurons. Collectively these findings suggest that interstitial cells are required for the normal development or maintenance of vagal intramuscular arrays. Therefore, the c-Kit mutant mouse will be valuable for determining the role(s) of interstitial cells in intramuscular array development as well as for providing an animal model with the intramuscular array class of vagal afferents selectively ablated.

To explore the effects of aging on the vagal innervation of the gastrointestinal (GI) tract, male Fischer 344 rats at 3 and 24 months of age were injected in the left nodose ganglion with 3 microl of either 4% wheat germ agglutinin-horseradish peroxidase (to label sensory endings) or 1% cholera toxin subunit B-horseradish peroxidase (to label motor endings). The stomach and duodenum were prepared as wholemounts and processed with tetramethyl benzidine. In addition, to study age-related changes in the myenteric plexus, the stomachs, small intestines, and large intestines from 3-, 12-, 21-, 24- and 27-month-old rats were prepared as wholemounts and processed with Cuprolinic Blue (to stain the neurons). Vagal afferent endings, motor terminal profiles, and myenteric neurons were counted and mapped with a sampling grid. In the stomach, both the vagal and myenteric innervation were stable between the ages of 3 and 24 months; however, a decrease in the number of myenteric neurons in the forestomach was noted at 27 months. In the small and large intestines, myenteric cell loss occurred by 12 months of age, progressed with age, and appeared to be governed by several general principles: (1) the rate of cell loss was organ-specific, with a gradient of increasing severity from proximal to distal in the gut; (2) within organs of the GI tract, the rate of cell loss differed between regions; and (3) for given regions, cell losses progressed linearly with increasing age. The findings suggest that a positive relationship exists between the density of vagal extrinsic innervation and myenteric neuron survival; however, whether this results from the vagal innervation and/or other factor(s) protecting or rescuing myenteric neurons from age-related attrition remains to be determined.

Intraganglionic laminar endings (IGLEs) and intramuscular arrays (IMAs), the two putative mechanoreceptors that the vagus nerve supplies to the gastrointestinal smooth muscle, have been characterized almost exclusively in the rat. To provide normative inventories of these afferents for the mouse, the authors examined the endings in the stomach and small intestine of three strains used as backgrounds for gene manipulations (i.e., C57, 129/SvJ, and WBB6). Animals received nodose ganglion injections of wheat germ agglutinin-horseradish peroxidase or dextran-tetramethylrhodamine conjugated to biotin. The horseradish peroxidase tissue was processed with tetramethylbenzidine and was used to map the distributions and densities of the two endings; the dextran material was counterstained with c-Kit immunohistochemistry to assess interactions between intramuscular arrays and interstitial cells of Cajal. IGLEs and IMAs constituted the vagal innervation of mouse gastric and duodenal smooth muscle. IGLE morphology and distributions, with peak densities in the corpus-antrum, were similar in the three strains of mice and comparable to those observed in rats. IMAs varied in complexity from region to region but tended to be simpler (fewer telodendria) in mice than in rats. IMAs were most concentrated in the forestomach and sphincters in mice, as in rats, but the topographic distributions of the endings varied both between strains of mice (subtly) and between species (more dramatically). IMAs appeared to make appositions with both interstitial cells and smooth muscle fibers. This survey should make it practical to assay the effects of genetic (e.g., knockout) and experimental (e.g., regeneration) manipulations affecting visceral afferents and their target tissues., (Copyright 2000 Wiley-Liss, Inc.)

Electrophysiological and morphological analyses of vagal mechanoreceptors in the gut wall suggest conflicting conclusions. Electrophysiology has distinguished a single general class of ending in smooth muscle, one characterized as an 'in series' tension receptor. Morphology, in contrast, has characterized two distinct specializations of vagal afferent endings in the muscle wall of the gastrointestinal (GI) tract. These two structures differ in terms of their target tissues, terminal architectures and regional distributions; they also develop on different ontogenetic timetables and depend on different trophic support in the muscle wall. On the basis of these features, we have proposed that one of the putative mechanoreceptors, the intraganglionic laminar ending (IGLE), has characteristics of a tension receptor and the other, the intramuscular array (IMA), has features of a stretch or length receptor. In a functional analogy with striated muscle proprioceptors, IGLEs should have similarities to Golgi tendon organs, whereas IMAs should have equivalencies with muscle spindle afferents. The present survey re-examines the recording analyses in light of the structural observations. This review indicates that previous electrophysiological studies are too inconclusive to refute the inference that the vagus supplies two distinct types of mechanoreceptors to the muscle wall of the GI tract. Multiple methodological constraints and sources of variance have limited the resolution of electrophysiological experiments. Specifically, these experiments have conventionally used distension stimuli that confound tension and stretch. In addition, sampling strategies have biased recording experiments towards a focus on one type of ending, the IGLE. Furthermore, putative functional properties (e.g., broad tuning) of vagal mechanoreceptors suggest that distinguishing two recording patterns will require exacting protocols. Combining a recognition of the methodological difficulties that have limited electrophysiological analyses with an understanding of the structural features of the endings, however, suggests several critical electrophysiological experiments with the resolution to distinguish two classes of response profiles. Until such experiments can be conducted, sensory physiology's axiom that 'function varies with form', taken together with a re-assessment of the existing data, suggests that the vagus nerve supplies stretch receptors as well as tension receptors to the wall of the GI tract.

We injected the anterograde fluorescent tracer 1,1;-dioleyl-3,3,3;, 3;-tetramethylindocarbocyanine methanesulfonate (DiI) into the nucleus ambiguus (NA) and used confocal microscopy to inventory NA fibers and axon terminals in whole-mounts of rat atrial tissues. Both the axons projecting to cardiac ganglia and the innervated principal neurons (PNs) were counted. Rats were injected unilaterally in the NA with DiI, either at four sites (between 600 microm rostral and 600 microm caudal to the obex) or at nine sites (1,600 microm rostral to 1,600 microm caudal). Fluoro-Gold was administered intraperitoneally to retrogradely label neurons of the dorsal motor nucleus of the vagus (DmnX), NA, and cardiac ganglia. To verify that the DiI-labeled fibers examined in the atria originated exclusively from the NA, neurons of the DmnX and the nodose ganglia were surveyed for DiI labeling. Our observations established that (1) NA fibers in the cardiac branches of the vagus numbered in the range of 82-151, left; or 60-122, right. (2) Both left and right NA supplied substantial numbers of fibers to each of the three major cardiac ganglionic plexuses. (3) NA axons terminated in dense basket, or calyx, endings around individual PNs. (4) By issuing divergent collaterals, individual NA fibers supplied numerous PNs with these calyx endings. (5) Labeled axons innervated 2,248 (left vagus) and 1,784 (right), or at least 56% and 45%, of the cardiac PNs. (6) Divergence (i.e., NA axons:PNs innervated) averaged between 1:27 (left vagus) and 1:30 (right vagus). Several features of these NA projections to cardiac ganglia contrasted sharply with those of DmnX projections that we have recently characterized with the same tracing protocol: (1) NA fibers did not innervate small intensely fluorescent cell clusters in cardiac ganglia, whereas DmnX axons did. (2) NA efferent fascicles contained more large fibers (presumably B-type), whereas the DmnX issued more fine caliber fibers (presumably C-type). (3) NA fibers diverged about three times as extensively as did DmnX axons. Taken together, our data strongly suggest that vagal control of the heart involves the convergence and integration of distinct NA and DmnX projections within the cardiac plexuses., (Copyright 2000 Wiley-Liss, Inc.)

To inventory and characterize the two types of vagal afferents (both putative mechanoreceptors) in the muscle of the gastrointestinal tract, the authors injected wheat germ agglutinin-horseradish peroxidase into the nodose ganglia of rats that had received unilateral ventral rhizotomies to eliminate efferents. The gut, from the oral esophagus to the distal colon, was divided into wholemounts, processed with tetramethylbenzidine, and surveyed to establish normative topographic maps of afferents. Vagal intraganglionic laminar endings (IGLEs) were ubiquitous, with concentrations varying on a longitudinal gradient (higher rostrally). This overall gradient was punctuated by denser condensations of endings in the oral esophagus, gastric corpus, and distal ileum. In regional specializations, IGLEs were fused into conspicuous, dense networks in the laryngeal esophagus and the antrum. Intramuscular arrays (IMAs) had restricted distributions, including the walls of the stomach and the sphincters throughout the gut. In the forestomach, a singular concentration of orthogonally crossed IMAs was organized into a lattice or "fovea." IMAs displayed variations in morphology, with one specialization consisting of short, terminal processes associated with sphincters and a more widespread form consisting of long, rectilinear processes in the forestomach, along the greater curvature, and in limited intestinal regions. On the basis of their topographic patterns and structural specializations, the two putative mechanoreceptors may have different functions: IGLEs appear situated to integrate intramural tension, and perhaps myenteric neuronal activity, into rhythmical, propagated motor programs, such as swallowing, peristalsis, and emptying. IMAs are distributed strategically and appear to satisfy structural requirements for stretch receptors tuned to tonic or more aperiodic events that may affect central nervous system processing as well as local gastrointestinal coordination., (Copyright 2000 Wiley-Liss, Inc.)

Peripheral projections of the vagus are known to regenerate after subdiaphragmatic vagotomy, but neither the question of whether the regenerating axons are motor or sensory nor the issue of whether the fibers reinnervate their original targets have been addressed. To determine whether vagal afferents regenerate and whether they differentiate into normal terminal specializations in the reinnervated target organ, male Sprague-Dawley rats underwent complete subdiaphragmatic vagotomies and were injected 18 weeks later with 3 microl of 4% wheat germ agglutinin-horseradish peroxidase (WGA-HRP) in the left nodose ganglion. To provide a comparison group, an unoperated group (controls) was injected with WGA-HRP in the left nodose ganglion. The esophagus, the entire stomach, the first 8 cm of the duodenum, and the hilus of the liver were prepared as wholemounts and processed with tetramethyl benzidine. Vagal afferents were found to have regenerated and reinnervated the esophagus, stomach, duodenum, and liver. Bundles (two or more axons), individual vagal axons, and terminals in the stomach were counted and mapped with a sampling grid. At 18 weeks postvagotomy, the reinnervated stomach and duodenum contained normal terminals as well as aberrant endings and growth cone profiles. The ingrowing axons reestablished ipsilateral and contralateral projections in the same proportions seen in controls, although the overall density of the different regenerating elements had reached only 7-39% of control values. These findings demonstrate that the gastrointestinal tract and liver can undergo dramatic afferent reinnervation after vagotomy. The presence of differentiated endings at 18 weeks suggests that some afferent function(s) may be restored, and the expression of growth cones suggests that additional regeneration may be ongoing., (Copyright 2000 Wiley-Liss, Inc.)

The dorsal vagal complex in the medulla oblongata is the hub of the central nervous system network that produces vagal cephalic-phase reflexes. The preganglionic motor neurons controlling these cephalic responses of digestion and metabolism are organized topographically in longitudinal columnar subnuclei in the dorsal motor nucleus of the vagus. Gustatory and other visceral afferent inputs project into different subnuclei of the nucleus of the solitary tract capping the dorsal motor nucleus. Descending projections from more rostral stations of the neuroaxis project to the nuclei of the dorsal vagal complex, providing input both from exteroceptive senses, such as olfaction and vision, and from forebrain areas that modulate reflex strength. Recent structural analyses of the dorsal vagal complex, as well as characterizations of the region's inputs and neurochemistry, have provided a more complete understanding of the neural basis of cephalic-phase responses., (Copyright 2000 Academic Press.)

We injected the anterograde fluorescent tracer 1,1'-dioleyl-3,3,3',3'-tetramethylindocarbocyanine methanesulfonate (DiI) into the dorsal motor nucleus of the vagus (DmnX), counterstained the cardiac ganglia with Fluorogold (FG), and used confocal microscopy to examine the distributions and different types of DmnX fibers in wholemounts of the atria. We also quantified the number of DmnX cardiac axons and the number of innervated cardiac principal neurons (PNs). Rats with unilateral DiI injections were used in three different experiments, including unilateral FG soaking of cervical vagal trunks, intracranially rhizotomizing the vagal afferent roots, or contralaterally sectioning the cervical vagus. These manipulations indicated that DiI-labeled cardiac fibers were exclusively from the DmnX. Our observations established that: (1) three major ganglionic plexuses were localized in the epicardium; (2) both sides of the DmnX supplied significant fibers to each of the plexuses; (3) these cardiac efferents formed dense basket terminals around individual PNs; (4) collaterals of individual DmnX fibers diverged, producing calyx endings on multiple PNs; (5) small intensely fluorescent (SIF) cells in the cardiac plexuses were innervated pericellularly; (6) individual axons could innervate both PNs and SIF cells; (7) the total number of DmnX fibers were in the range of [68, 96; left] and [67, 115; right]; (8) these fibers innervated 709 (left) and 494 (right), or at least 18% and 12%, of the PNs, respectively; and (9) vagal preganglionics exhibited a degree of lateralization: Significantly more PNs were contacted by fiber varicosities in the sinoatrial plexus than in the atrioventricular plexus after right DmnX injections. In summary, the present observations suggest that the DmnX plays a significant role(s) in controlling the heart.

Nitrergic (NO) neurons play crucial inhibitory roles in the control of gut motility. Variations in the density of these neurons within the gastrointestinal tract (GI) may provide useful functional information, but, most surveys available have employed limited and/or highly localized samples. It remains unclear to what extent (a) NO neurons are concentrated disproportionately in particular GI regions, or (b) variations in NO cell number merely reflect changes in overall myenteric neuron density. This experiment surveyed the distributions of neuronal nitric oxide synthase-positive (NOS+) and other myenteric neurons in the GI tract, using immunohistochemical and Cuprolinic blue counterstaining techniques. Adjustable sampling grids superimposed on wholemounts were used to investigate the topographic patterns in the stomach (90 sampling sites; 45 per side) and proximal duodenum (63 loci). We present four major findings: First, variations were detected in the number of NOS+ neurons in specific regions of the stomach (e.g., corpus > antrum approximately equal to forestomach) and along both longitudinal (oral > anal) and circumferential (mesenteric > antimesenteric) axes in the duodenum. Second, the variations in NOS+ neuronal counts within each organ covaried with the total number of myenteric neurons at different locations (stomach, r=0.77; duodenum, r=0.59), suggesting that local myenteric plexus density is a factor determining NOS+ cell concentrations. Third, in contrast to such a principle of covariation within each organ, NOS+ neurons constituted a consistently smaller proportion of gastric (20%) than of duodenal (28%) myenteric plexus neurons, suggesting that a second principle controls the characteristic percentages of the myenteric plexus that express NOS in different organs. Fourth, the regional samples were used to extrapolate the overall number of NOS+ and total myenteric cells in the rat stomach (43,000; 217,000) and first 3.5 cm of the small intestine (29,000; 103,000). These results, taken together, also suggest that the surveying protocol used is capable of detecting subtle differences in cellular distributions, thus providing a practical strategy for investigating patterns of chemical phenotypes within the GI tract.

The dorsal motor nucleus of the vagus (DMNX) contains neurons with different projections and discrete functions, but little success has been achieved in distinguishing the cells cytoarchitectonically. The present experiment employed multivariate analytical techniques to evaluate DMNX neuronal morphology. Male Sprague-Dawley rats (n = 77) were perfused, and the brainstems were stained en bloc with a Golgi-Cox protocol. DMNX neurons in each of three planes (coronal, sagittal, and horizontal; total sample = 607) were digitized. Three-dimensional features quantified included dendritic length, number of segments, spine density, number of primary dendrites, dendritic orientation, and soma form factor. Cluster analyses of six independent samples of 100+ neurons and of three composite replicate pools of 200+ neurons consistently identified similar sets of four distinct neuronal profiles. One profile (spinous, limited dendrites, small somata) appears to correspond to the interneuron population of the DMNX. In contrast, the other three distinctive profiles (e.g., one is multipolar, with large dendritic fields and large somata) are different types of preganglionic neurons. Each of the four types of neurons is found throughout the DMNX, suggesting that the individual columnar subnuclei and other postulated vagal motorneuron pools are composed of all types of neurons. Within individual motor pools, ensembles of the different neuronal types must cooperatively organize different functions and project to different effectors within a target organ. By extension, specializations of the preganglionic motor pools are more likely to result from their afferent inputs, peripheral target tissues, neurochemistry, or physiological features rather than from any unique morphological profiles.

Rats receiving intragastric infusions of 2.5, 5.0, 7.5, or 10.0 ml of normal saline while their pylori are reversibly occluded suppress meal size to the smallest infusion and display a dose-dependent reduction across volumes [Phillips, R. J., and T. L. Powley. Am. J. Physiol. 271 (Regulatory Integrative Comp. Physiol. 40): R766-R779, 1996]. To evaluate the contributions of the vagus to this detection of gastric volume, groups prepared with different selective vagotomies and equipped with pyloric cuffs and gastric catheters were tested. Liquid diet consumption during a 30-min feeding bout was measured after infusions of 5.0 and 10.0 ml of normal saline on cuff-open and cuff-closed trials. Consistent with earlier observations, sham animals with cuffs closed exhibited volume-dependent suppression of food intake to the infusions, and completely vagotomized animals did not inhibit feeding in response to the loads. In cuff-closed trials, the suppression function slopes of the selective vagotomy groups were intermediate to those of the shams and the completely vagotomized animals. Furthermore, for the different groups, the extent of suppression after vagotomy was proportional to the density of the afferent innervation respective branches supplied to the stomach. Specifically, the group with the gastric branches spared (nonsignificantly attenuated in comparison to shams) and the group with only the hepatic branch spared (significantly attenuated with respect to shams) both still exhibited significant dose-dependent suppression slopes (compared with completes), whereas the group with only celiac branches spared was not significantly different from completely vagotomized animals. In sum, the vagus nerve mediates the detection of the gastric volumes tested, and the different branches of the vagus make distinctive contributions to this afferent feedback.

Potential effects of environmental rearing conditions on the brains of farm animals have not been examined experimentally, with the exception of one report for pig somatosensory cortex. The goal of the present experiment was to determine whether different developmental environments in use in agricultural production units affect neuronal morphology in the pig cerebral cortex. Littermate female pigs (gilts) were cross-fostered at birth and reared in either an indoor (n = 6) or outdoor (n = 6) production unit for 8 weeks. Additional littermates (n = 6) were sacrificed at 3 days of age to provide a developmental reference point. Brains were fixed by perfusion and stained by the Golgi-Cox method. The primary somatosensory, auditory and visual cortices were sectioned at 170 microns, and layer IV stellate neurons (n = 492) were digitized and 3-dimensionally reconstructed. Measurements of dendritic length, membrane surface area, total number of segments, number of 1st- through 7th-order dendrites, spine density, soma area, and soma form factor were taken. In auditory cortex neurons, outdoor pigs compared to indoor pigs had (a) significantly more primary dendrites, (b) significantly greater spine density, and (c) trends of increases both in number of 2nd- and 3rd-order dendrites and in total dendritic length. In visual cortex neurons, indoor pigs had significantly more 7th-order dendrites, whereas in all three cortical areas, the indoor animals had more 5th-order dendrites. Multiple morphological differences occurred in stellate cell populations between the three sensory areas of the Week 8 pigs. Also, within different cortical areas, dendritic morphology changed substantially from 3 days to 8 weeks of age. Further investigations are needed to determine which environmental factors are critical in producing the observed changes in brain morphology and whether other brain effects may be produced by varying developmental environments.

Although the aortic nerves contain vagal afferents that terminate in both the wall of the aortic arch (putative baroreceptors) and its associated glomus tissue (putative chemoreceptors) in most mammalian species, the aortic nerves of the rat have been widely assumed to contain only baro- or pressor afferents. The present study reconsidered this anomaly by characterizing vagal afferent endings and their targets in the aortic arch region of the rat, both qualitatively and quantitatively. Eight Sprague-Dawley rats received intracranial vagal motor rhizotomy unilaterally to eliminate efferents in the nerve and then, two weeks later, injections of the tracer DiI (1,1'-dioleyl-3,3,3',3'-tetramethylindocarbocyanine methanesulfonate) into the ipsilateral nodose ganglion. The aortic arch and its surrounding tissue, with the common carotid and subclavian arteries attached, were examined with both conventional epifluorescence and confocal microscopes. Consistent with earlier observations, vagal afferents formed both flower-spray and end-net terminals rather diffusely within the wall of the aortic arch. More interestingly, vagal afferents also innervated glomus or SIF (i.e., small intensely fluorescent) cell bodies at the junction areas of the common carotid and subclavian arteries. To identify the course of these fibers, six additional animals received DiI injection into the nodose unilaterally after a complete cervical vagotomy caudal to the nodose; in these animals, the aortic nerve had been separated from the vagal trunk and kept intact. There were no marked differences in innervation patterns between the nonvagotomized and the cervically vagotomized animals, indicating that the vagal axons innervating the walls of the blood vessels and the SIF cells in the aortic arch region travel through the aortic nerves. Using a stereological method, we estimated the relative number of chemo- and baroreceptor afferents innervating the aortic arch. About 16.4% (left) and 13.1% (right) of fibers in the aortic nerves innervate SIF cells. These findings challenge the general consensus that the aortic nerves of rats contain exclusively baroreceptor fibers.

Experiment 1 used Pavlovian conditioning procedures to show that rats formed distinct memorial representations of 2 (peanut oil and sucrose pellets) unconditioned stimuli (USs) that could be activated by 2 different conditioned stimuli (CSs). After training in Experiment 2, rats injected with the lipid antimetabolite Na-2-mercaptoacetate (MA) responded more to the CS for oil than to the CS for sucrose. This pattern was not shown by rats that received isotonic saline or systemic 2-deoxy-d-glucose (a glucose antimetabolite). By contrast, intracerebroventricular infusion of the glucose antimetabolite 5-thioglucose selectively promoted responding to the CS for sucrose (Experiment 4). Thus, lipoprivic and glucoprivic treatments selectively promoted the activation of the memories of fat and carbohydrate USs, respectively. In Experiment 3, the capacity of MA to augment responding to a CS for oil was abolished for rats that received subdiaphragmatic vagal deafferentation. This indicates that the capacity of lipoprivic signals to selectively activate the representations of fat USs may depend on vagal afferent fibers.