Stomach region stimulated determines effects on duodenal motility in rats.

Gastric electrical stimulation (GES) is used clinically to promote proximal GI emptying and motility. In acute experiments, we measured duodenal motor responses elicited by GES applied at 141 randomly chosen electrode sites on the stomach serosal surface. Overnight-fasted (H2O available) anesthetized male rats (n = 81) received intermittent biphasic GES for 5 min (20-s-on/40-s-off cycles; I = 0.3 mA; pw = 0.2 ms; 10 Hz). A strain gauge on the serosal surface of the proximal duodenum of each animal was used to evaluate baseline motor activity and the effect of GES. Using ratios of time blocks compared with a 15-min prestimulation baseline, we evaluated the effects of the 5-min stimulation on concurrent activity, on the 10 min immediately after the stimulation, and on the 15-min period beginning with the onset of stimulation. We mapped the magnitude of the duodenal response (three different motility indices) elicited from the 141 stomach sites. Post hoc electrode site maps associated with duodenal responses suggested three zones similar to the classic regions of forestomach, corpus, and antrum. Maximal excitatory duodenal motor responses were elicited from forestomach sites, whereas inhibitory responses occurred with stimulation of the corpus. Moderate excitatory duodenal responses occurred with stimulation of the antrum. Complex, weak inhibitory/excitatory responses were produced by stimulation at boundaries between stomach regions. Patterns of GES efficacies coincided with distributions of previously mapped vagal afferents, suggesting that excitation of the duodenum is strongest when GES electrodes are situated over stomach concentrations of vagal intramuscular arrays, putative stretch receptors in the muscle wall.

Carbohydrates designed with different digestion rates modulate gastric emptying response in rats.

We sought to determine whether design of carbohydrate-based microspheres to have different digestion rates, while retaining the same material properties, could modulate gastric emptying through the ileal brake. Microspheres made to have three slow digestion rates and a rapidly digested starch analogue (maltodextrin) were administrated to rats by gavage and starch contents in the stomach, proximal and distal small intestine, and caecum were measured 2 h post-gavage. A stepwise increase in the amount of starch retained in the stomach was found for microspheres with incrementally slower rates of digestion. Postprandial glycaemic and insulinaemic responses were incrementally lower for the different microspheres than for the rapidly digestible control. A second-meal effect was observed for slowly digestible starch (SDS) microspheres compared to glucose. Thus, dietary slowly digestible carbohydrates were designed to elicit incremental significant changes in gastric emptying, glycaemic and insulinaemic responses, and they may be a means to trigger the ileal brake.

Gastric stimulation drives fast BOLD responses of neural origin.

Functional magnetic resonance imaging (fMRI) is commonly thought to be too slow to capture any neural dynamics faster than 0.1 Hz. However, recent findings demonstrate the feasibility of detecting fMRI activity at higher frequencies beyond 0.2 Hz. The origin, reliability, and generalizability of fast fMRI responses are still under debate and await confirmation through animal experiments with fMRI and invasive electrophysiology. Here, we acquired single-echo and multi-echo fMRI, as well as local field potentials, from anesthetized rat brains given gastric electrical stimulation modulated at 0.2, 0.4 and 0.8 Hz. Such gastric stimuli could drive widespread fMRI responses at corresponding frequencies from the somatosensory and cingulate cortices. Such fast fMRI responses were linearly dependent on echo times and thus indicative of blood oxygenation level dependent nature (BOLD). Local field potentials recorded during the same gastric stimuli revealed transient and phase-locked broadband neural responses, preceding the fMRI responses by as short as 0.5 s. Taken together, these results suggest that gastric stimulation can drive widespread and rapid fMRI responses of BOLD and neural origin, lending support to the feasibility of using fMRI to detect rapid changes in neural activity up to 0.8 Hz under visceral stimulation.

Brain-to-stomach transfer of α-synuclein via vagal preganglionic projections.

Detection of α-synuclein lesions in peripheral tissues is a feature of human synucleinopathies of likely pathogenetic relevance and bearing important clinical implications. Experiments were carried out to elucidate the relationship between α-synuclein accumulation in the brain and in peripheral organs, and to identify potential pathways involved in long-distance protein transfer. Results of this in vivo study revealed a route-specific transmission of α-synuclein from the rat brain to the stomach. Following targeted midbrain overexpression of human α-synuclein, the exogenous protein was capable of reaching the gastric wall where it was accumulated into preganglionic vagal terminals. This brain-to-stomach connection likely involved intra- and inter-neuronal transfer of non-fibrillar α-synuclein that first reached the medulla oblongata, then gained access into cholinergic neurons of the dorsal motor nucleus of the vagus nerve and finally traveled via efferent fibers of these neurons contained within the vagus nerve. Data also showed a particular propensity of vagal motor neurons and efferents to accrue α-synuclein and deliver it to peripheral tissues; indeed, following its midbrain overexpression, human α-synuclein was detected within gastric nerve endings of visceromotor but not viscerosensory vagal projections. Thus, the dorsal motor nucleus of the vagus nerve represents a key relay center for central-to-peripheral α-synuclein transmission, and efferent vagal fibers may act as unique conduits for protein transfer. The presence of α-synuclein in peripheral tissues could reflect, at least in some synucleinopathy patients, an ongoing pathological process that originates within the brain and, from there, reaches distant organs innervated by motor vagal projections.

Prostaglandin pathway gene expression in human placenta, amnion and choriodecidua is differentially affected by preterm and term labour and by uterine inflammation.

BACKGROUND: Elucidation of the biochemical pathways involved in activation of preterm and term human labour would facilitate the development of effective management and inform judgements regarding the necessity for preterm tocolysis and post-term induction. Prostaglandins act at all stages of human reproduction, and are potentially activators of labour. METHODS: Expression of 15 genes involved in prostaglandin synthesis, transport and degradation was measured by qPCR using tissue samples from human placenta, amnion and choriodecidua at preterm and full-term vaginal and caesarean delivery. Cellular localisation of eight prostaglandin pathway proteins was determined by immunohistochemistry. RESULTS: Expression of prostaglandin pathway genes was differentially affected by factors including gestational age at delivery, and the incidence and duration of labour. Chorioamnionitis/deciduitis was associated with upregulation of PTGS2 (prostaglandin-endoperoxide synthase 2 (prostaglandin G/H synthase and cyclooxygenase)), along with the inflammatory genes IL8 (interleukin 8), S100A8 (S100 calcium binding protein A8) and TLR2 (toll-like receptor 2), in amnion and choriodecidua, and with downregulation of CBR1 (carbonyl reductase 1) and HPGD (hydroxyprostaglandin dehydrogenase 15-(NAD)) in choriodecidua. Protein localisation differed greatly between the various maternal and fetal cell types. CONCLUSIONS: Preterm and term labour are associated with distinct prostaglandin pathway expression profiles; inflammation provokes specific changes, unrelated to the presence of labour; spontaneous and induced term labour are indistinguishable.

Versatile, high-resolution anterograde labeling of vagal efferent projections with dextran amines.

None of the anterograde tracers used to label and investigate vagal preganglionic neurons projecting to the viscera has proved optimal for routine and extensive labeling of autonomic terminal fields. To identify an alternative tracer protocol, the present experiment evaluated whether dextran conjugates, which have produced superior results in the CNS, might yield widespread and effective labeling of long, fine-caliber vagal efferents in the peripheral nervous system. The dextran conjugates that were evaluated proved reliable and versatile for labeling the motor neuron pool in its entirety, for single- and multiple-labeling protocols, for both conventional and confocal fluorescence microscopy, and for permanent labeling protocols for brightfield microscopy of the projections to the gastrointestinal (GI) tract. Using a standard ABC kit followed by visualization with DAB as the chromagen, Golgi-like labeling of the vagal efferent terminal fields in the GI wall was achieved with the biotinylated dextrans. The definition of individual terminal varicosities was so sharp and detailed that it was routinely practical to examine the relationship of putative vagal efferent contacts (by the criteria of high magnification light microscopy) with the dendritic and somatic architecture of counterstained neurons in the myenteric plexus. Overall, dextran conjugates provide high-definition labeling of an extensive vagal motor pool in the GI tract, and offer considerable versatility when multiple-staining protocols are needed to elucidate the complexities of the innervation of the gut.

Vagal innervation of the stomach reassessed: brain-gut connectome uses smart terminals.

Brain-gut neural communications have long been considered limited because of conspicuous numerical mismatches. The vagus, the parasympathetic nerve connecting brain and gut, contains thousands of axons, whereas the gastrointestinal (GI) tract contains millions of intrinsic neurons in local plexuses. The numerical paradox was initially recognized in terms of efferent projections, but the number of afferents, which comprise the majority (≈ 80%) of neurites in the vagus, is also relatively small. The present survey of recent morphological observations suggests that vagal terminals, and more generally autonomic and visceral afferent arbors in the stomach as well as throughout the gut, elaborate arbors that are extensive, regionally specialized, polymorphic, polytopic, and polymodal, commonly with multiplicities of receptors and binding sites-smart terminals. The morphological specializations and dynamic tuning of one-to-many efferent projections and many-to-one convergences of contacts onto afferents create a complex architecture capable of extensive peripheral integration in the brain-gut connectome and offset many of the disparities between axon and target numbers. Appreciating this complex architecture can help in the design of therapies for GI disorders.

Dietary Slowly Digestible Starch Triggers the Gut-Brain Axis in Obese Rats with Accompanied Reduced Food Intake.

SCOPE: Slowly digestible starch (SDS), as a functional carbohydrate providing a slow and sustained glucose release, may be able to modulate food intake through activation of the gut-brain axis. METHODS AND RESULTS: Diet-induced obese rats were used to test the effect on feeding behavior of high-fat (HF) diets containing an SDS, fabricated to digest into the ileum, as compared to rapidly digestible starch (RDS). Ingestion of the HF-SDS diet over an 11-week period reduced daily food intake, through smaller meal size, to the same level as a lean body control group, while the group consuming the HF-RDS diet remained at a high food intake. Expression levels (mRNA) of the hypothalamic orexigenic neuropeptide Y (NPY) and Agouti-related peptide (AgRP) were significantly reduced, and the anorexigenic corticotropin-releasing hormone (CRH) was increased, in the HF-SDS fed group compared to the HF-RDS group, and to the level of the lean control group. CONCLUSION: SDS with digestion into the ileum reduced daily food intake and paralleled suppressed expression of appetite-stimulating neuropeptide genes associated with the gut-brain axis. This novel finding suggests further exploration involving a clinical study and potential development of SDS-based functional foods as an approach to obesity control.

Innervation of the gastrointestinal tract: patterns of aging.

The gastrointestinal (GI) tract is innervated by intrinsic enteric neurons and by extrinsic projections, including sympathetic and parasympathetic efferents as well as visceral afferents, all of which are compromised by age to different degrees. In the present review, we summarize and illustrate key structural changes in the aging innervation of the gut, and suggest a provisional list of the general patterns of aging of the GI innervation. For example, age-related neuronal losses occur in both the myenteric plexus and submucosal plexus of the intestines. These losses start in adulthood, increase over the rest of the life span, and are specific to cholinergic neurons. Parallel losses of enteric glia also occur. The extent of neuronal and glial loss varies along an oral-to-anal gradient, with the more distal GI tract being more severely affected. Additionally, with aging, dystrophic axonal swellings and markedly dilated varicosities progressively accumulate in the sympathetic, vagal, dorsal root, and enteric nitrergic innervation of the gut. These dramatic and consistent patterns of neuropathy that characterize the aging autonomic nervous system of the GI tract are candidate mechanisms for some of the age-related declines in function evidenced in the elderly.

Effects of age on sympathetic innervation of the myenteric plexus and gastrointestinal smooth muscle of Fischer 344 rats.

Loss of myenteric neurons with age is well documented, however little is known about age-related changes of the sympathetic innervation of the myenteric plexus and gastrointestinal smooth muscle. The goal of the present study, therefore, was to evaluate the influence of age on the sympathetic innervation of the myenteric plexus throughout the gastrointestinal tract. Ad libitum fed virgin male Fischer 344 rats at 3, 15-16, 24, and 27-28 months of age were sampled. Whole mounts of the stomach, small intestine, and large intestine were processed with an antibody to tyrosine hydroxylase (TH). Additionally, some specimens labeled for TH were stained for NADPH-diaphorase to selectively label the nitrergic subpopulation of neurons in the myenteric plexus. Age-related changes in the TH-positive axons occurred as early as 15-16 months and became more pronounced by 27-28 months. Changes included markedly swollen axons and terminals and a decrease in the intensity of TH staining in some of the surviving processes. Similarly, swollen NADPH-diaphorase-positive axons were found in the myenteric ganglia and secondary plexus between ganglia in the whole mounts of rats 15-28 months of age, but swollen nitrergic axons and dystrophic TH-positive axons were never present in the same ganglion or connective. Therefore, in the aged rat, deterioration of the sympathetic innervation of the myenteric plexus could be one possible mechanism for the age-related decline in gastrointestinal motor function evidenced in the elderly.

Individual sympathetic postganglionic neurons coinnervate myenteric ganglia and smooth muscle layers in the gastrointestinal tract of the rat.

A full description of the terminal architecture of sympathetic axons innervating the gastrointestinal (GI) tract has not been available. To label sympathetic fibers projecting to the gut muscle wall, dextran biotin was injected into the celiac and superior mesenteric ganglia (CSMG) of rats. Nine days postinjection, animals were euthanized and stomachs and small intestines were processed as whole mounts (submucosa and mucosa removed) to examine CSMG efferent terminals. Myenteric neurons were counterstained with Cuprolinic Blue; catecholaminergic axons were stained immunohistochemically for tyrosine hydroxylase. Essentially all dextran-labeled axons (135 of 136 sampled) were tyrosine hydroxylase-positive. Complete postganglionic arbors (n = 154) in the muscle wall were digitized and analyzed morphometrically. Individual sympathetic axons formed complex arbors of varicose neurites within myenteric ganglia/primary plexus and, concomitantly, long rectilinear arrays of neurites within circular muscle/secondary plexus or longitudinal muscle/tertiary plexus. Very few CSMG neurons projected exclusively (i.e., ∼100% of an arbor's varicose branches) to myenteric plexus (∼2%) or smooth muscle (∼14%). With less stringent inclusion criteria (i.e., ≥85% of an axon's varicose branches), larger minorities of neurons projected predominantly to either myenteric plexus (∼13%) or smooth muscle (∼27%). The majority (i.e., ∼60%) of all individual CSMG postganglionics formed mixed, heterotypic arbors that coinnervated extensively (>15% of their varicose branches per target) both myenteric ganglia and smooth muscle. The fact that ∼87% of all sympathetics projected either extensively or even predominantly to smooth muscle, while simultaneously contacting myenteric plexus, is consistent with the view that these neurons control GI muscle directly, if not exclusively. J. Comp. Neurol. 524:2577-2603, 2016. © 2016 Wiley Periodicals, Inc.

Vagal Intramuscular Arrays: The Specialized Mechanoreceptor Arbors That Innervate the Smooth Muscle Layers of the Stomach Examined in the Rat.

The fundamental roles that the stomach plays in ingestion and digestion notwithstanding, little morphological information is available on vagal intramuscular arrays (IMAs), the afferents that innervate gastric smooth muscle. To characterize IMAs better, rats were given injections of dextran biotin in the nodose ganglia, and, after tracer transport, stomach whole mounts were collected. Specimens were processed for avidin-biotin permanent labeling, and subsets of the whole mounts were immunohistochemically processed for c-Kit or stained with cuprolinic blue. IMAs (n = 184) were digitized for morphometry and mapping. Throughout the gastric muscle wall, IMAs possessed common phenotypic features. Each IMA was generated by a parent neurite arborizing extensively, forming an array of multiple (mean = 212) branches averaging 193 µm in length. These branches paralleled, and coursed in apposition with, bundles of muscle fibers and interstitial cells of Cajal. Individual arrays averaged 4.3 mm in length and innervated volumes of muscle sheet, presumptive receptive fields, averaging 0.1 mm(3) . Evaluated by region and by muscle sheet, IMAs displayed architectural adaptations to the different loci. A subset (32%) of circular muscle IMAs issued specialized polymorphic collaterals to myenteric ganglia, and a subset (41%) of antral longitudinal muscle IMAs formed specialized net endings associated with the serosal boundary. IMAs were concentrated in regional patterns that correlated with the unique biomechanical adaptations of the stomach, specifically proximal stomach reservoir functions and antral emptying operations. Overall, the structural adaptations and distributions of the IMAs were consonant with the hypothesized stretch receptor roles of the afferents.

Regulation of expression of the chorionic gonadotropin/luteinizing hormone receptor gene in the human myometrium: involvement of specificity protein-1 (Sp1), Sp3, Sp4, Sp-like proteins, and histone deacetylases.

At present there is little information on the regulatory processes by which the chorionic gonadotropin (CG)/LH receptor gene is regulated in the human myometrium during pregnancy and labor. Employing human primary myometrial cell cultures in conjunction with DNA affinity purification assays/Western analysis, DNA binding studies, CG/LH promoter luciferase reporter gene deletion constructs in transfection assays, and measurement of endogenous mRNA levels in vivo by duplex RT-PCR, we have determined the role that the major transcriptional regulatory sequences from the +1 ATG codon to -2678 bp play in modulating expression of the CG/LH receptor gene in the myometrium. We report that the distal -180 to -2678 bp region of the promoter, although capable of binding members of the Jun family via the multiple activator protein-1 sites within this region, has no significant role in regulating the expression of the CG/LH receptor gene in myometrial cells. In contrast, the two specificity protein-1 to -4 (Sp1-4) GC boxes within the +1 to -180 bp proximal promoter are central to expression of the gene in the myometrium. However, not only are Sp1/Sp3 proteins involved in this process, but Sp4 and a novel Sp-like factor(s) also have an intimate part in transcriptional regulation of the gene. It would appear that Sp1/Sp3/Sp4 and Sp-like proteins are involved in recruiting histone deacetylase complexes to the proximal promoter, preventing chromatin remodeling resulting in transcriptional repression of the gene. Our data suggest that administration of the histone deacetylase inhibitor trichostatin A to human myometrial cells in vitro and in vivo substantially removes this silencing effect on expression of the gene and may implicate the use of this and similar agents in increasing myometrial CG/LH receptor levels and subsequent maintenance of uterine relaxation during fetal maturation.

Obesity: should treatments target visceral afferents?

The fact that obesity is a chronic disorder has traditionally focused experimental attention on the long-term controls of energy balance. Searches for therapeutic targets tend to concentrate on central integrative mechanisms and to largely ignore the visceral afferents and other peripheral mechanisms providing short-term controls of energy balance. Investigations of central mechanisms have yet to yield, however, any practical and effective treatments for correcting obesity. In this review, we survey some of the arguments for considering peripheral visceral afferent mechanisms as promising targets for future research on obesity. These arguments include (1) the observation that visceral afferents have the specializations, complexities, heterogeneities, and extensive distributions at key sites to provide exhaustive and dynamic feedback to control energy handling, (2) the fact that the most effective treatments yet developed for achieving long-term or permanent weight loss, namely gastroplasty and similar bariatric surgical procedures, clearly alter visceral afferent feedback from the gastrointestinal tract, and (3) experimental observations that suggest loss of visceral negative feedback can lead to overeating, positive energy balance, and obesity. Furthermore, even though excess adiposity is a disturbance in long-term energy regulation, it is instructive that obesity in the final analysis is developed, is maintained, and ultimately needs to be treated one meal at a time. When these considerations are taken in conjunction with concerns about side effects and risks that can be expected to accompany pharmacological therapies directed at central nervous system circuits, it would seem prudent to assess ways in which the feedback of visceral afferents might be enhanced or manipulated to support or synergize with other therapeutic strategies used in the management of excess energy intake.

Plasticity of vagal afferents at the site of an incision in the wall of the stomach.

Our objectives were to determine whether the vagal afferent innervation of the stomach reorganizes after surgery and to observe how different wound closure techniques might influence such a process. The smooth muscle wall of the stomach served as a model because it is densely innervated by vagal axons and is frequently compromised by gastric surgery. Male Sprague-Dawley rats were assigned to one of six groups: three groups served as controls in which the stomach was exposed surgically and a) subjected to no further manipulation, b) traumatized with suture needle punctures of the muscle wall, or c) insulted by the placement of knotted suture thread in the stomach muscle; three surgical groups received a 1.0 cm incision through the ventral muscle wall of the stomach that was closed using either a) absorbable sutures, b) fibrin glue, or c) n-butyl cyanoacrylate. Rats were killed 4 to 7 months post-surgery. Prior to euthanasia, Micro-Ruby was injected into the left nodose ganglion of each rat to label vagal afferent axons and terminals. Twelve days post-injection, the stomachs were processed for microscopy. All groups recovered quickly from surgery, without differences in body weight. The presence of suture material in the muscle wall of the stomach was sufficient to produce reorganization of nearby vagal afferents. In addition, we observed that an incision of the smooth muscle wall of the stomach and the associated damage to vagal afferents provoked reorganization and regeneration of vagal afferents. Vagal remodeling at the incision was characteristic of axonal patterns found in neuromas (unlike the organized regeneration and differentiation that can occur after axotomy central to the target organ). Vagal afferent terminals located at the site of the incision were free nerve endings and growth cone profiles, and not the characteristically complex end organs normally found in the smooth muscle. Finally, the pattern of vagal plasticity was influenced by the wound closure technique used. Overall, the remodeling of afferents was aberrant in nature, and such neural pathology could contribute to the neuropathic symptoms and hyperalgesias associated with gastrointestinal trauma and bariatric surgery.

Differential expression of the adenylyl cyclase-stimulatory guanosine triphosphate-binding protein G(s)alpha in the human myometrium during pregnancy and labor involves transcriptional regulation by cyclic adenosine 3',5'-monophosphate and binding of phosphorylated nuclear proteins to multiple GC boxes within the promoter.

G(s)alpha is the G protein subunit that stimulates adenylyl cyclase activity in the myometrium during pregnancy, raising intracellular levels of the smooth muscle relaxant cAMP. The promoter region of the gene encoding G(s)alpha is GC rich and contains multiple putative binding sites for the specificity protein (Sp) transcription factor family. In electrophoretic mobility shift assays, four of these Sp sites were bound by recombinant Sp1 protein. Binding was dependent on phosphorylation of Sp1 by protein kinase A. Phosphorylated Sp1-4 proteins were observed in extracts of cultured human myometrial cells, but in electrophoretic mobility shift assays G(s)alpha promoter sequence binding by Sp1 was not apparent. Instead, these assays showed phosphorylation-dependent G(s)alpha promoter binding by lower molecular weight myometrial proteins that could not be supershifted by antibodies specific to Sp1-4 proteins. To investigate the regulation of G(s)alpha expression, the GC-rich promoter region was used to direct transcription of a firefly luciferase reporter gene in transient transfection assays of primary human myometrial cell cultures, COS-7 and HEK 293 cells. Reporter gene expression was found to follow a biphasic response to forskolin and 8-bromo-cAMP, with an initial, concentration-dependent increase in luciferase activity, followed by a prolonged decrease. In myometrial cells, this pattern was also seen in response to treatment with human chorionic gonadotropin.

Characterization and functional analysis of cAMP response element modulator protein and activating transcription factor 2 (ATF2) isoforms in the human myometrium during pregnancy and labor: identification of a novel ATF2 species with potent transactivation properties.

There is now extensive evidence to indicate that components of the cAMP signaling pathway are up-regulated in the human myometrium during pregnancy so as to potentiate the maintenance of uterine quiescence until term. In many tissue and cell types, increased signaling of the cAMP pathway results in profound changes in gene expression that are catalyzed via stimulation of PKA and activation of cAMP-dependent transcription factors that bind cAMP response elements (CREs) within the promoter regions of affected genes. In the myometrium, these CRE containing genes include beta2-adrenoceptor, cyclo-oxygenase 2, oxytocin receptor, and connexin-43. In preliminary investigations, we reported the differential expression of members of the cAMP bZIP protein family in the myometrium during pregnancy and labor. In this present study, we have now identified and functionally characterized these proteins with respect to myometrial gene expression. We report the identification of a 39,000 mol wt CRE response element modulator protein (CREM)tau2alpha protein having both transactivation and transrepressor properties whose expression is sequentially decreased in the myometrium during gestation and parturition. In contrast, expression of a myometrial 28,000 mol wt CREMalpha protein having only transrepressor actions progressively increased in the myometrium during pregnancy and labor. Similarly, we have isolated two ATF2 proteins of 60,000 and 28,000 mol wts, which represent full-length ATF2 and a novel small isoform of ATF2 that we have termed ATF2-small (ATF2-sm). These proteins are potent transactivators of gene expression and appear to be spatially expressed within the myometrium of the upper and lower uterine regions. The identification and functional characterization of these basic region/leucine zipper proteins in the myometrium may provide further insight into the molecular mechanisms regulating uterine activity during fetal maturation and parturition.

Long-term regeneration of abdominal vagus: efferents fail while afferents succeed.

Vagal afferents regenerate, by 18 weeks after subdiaphragmatic transection, to reinnervate the gut and to differentiate into the two types of terminals normally found in the smooth muscle wall of the gastrointestinal (GI) tract (Phillips et al. [2000] J Comp Neurol. 421:325-346). Regeneration, however, is neither complete nor entirely accurate by 18 weeks. Moreover, the capacity of the vagal efferents to reinnervate the GI tract under comparable conditions has not been evaluated. Therefore, to determine whether a more extended postaxotomy survival interval would (1). result in more extensive reinnervation of smooth muscle, (2). facilitate correction of the inaccuracies of the regenerated axons and terminals, and (3). yield motor as well as sensory reinnervation of GI targets, Sprague-Dawley rats received either complete subdiaphragmatic vagotomies (n = 18) or sham surgeries (n = 12). Physiological endpoints that might normalize as vagal elements regenerated, including body weight, daily food intake, size of first daily meal, and metabolic efficiency, were monitored. At 45 weeks after the vagotomies, the animals were randomly assigned to afferent (wheat germ agglutinin-horseradish peroxidase) or efferent (cholera toxin subunit B-horseradish peroxidase) mapping conditions, and labeled axons and terminals in the stomach and first 8 cm of the small intestine were inventoried in whole-mounts. Afferent regeneration was more extensive at 45 weeks than previously observed at 18 weeks after surgery; however, the amount of GI innervation was still not comparable to the intact pattern of the sham rats. Furthermore, abnormal patterns of sensory organization occurred throughout the reinnervated field, with small bundles of axons forming complex tangles and some individual axons terminating in ectopic locations. The presence of growth cone profiles suggested that vagal reorganization was ongoing even 45 weeks after surgery. In contrast to this relatively extensive, albeit incomplete, sensory reinnervation of the gut, motor fibers had failed to reinnervate the GI tract. Thus, dramatic differences exist in the regenerative capacities of the sensory and motor arms of the vagus under the same surgical and maintenance conditions. Furthermore, the functional measures of disordered energy regulation did not normalize over the 45 weeks during which afferent but not efferent innervation was restored.

Quantification of neurons in the myenteric plexus: an evaluation of putative pan-neuronal markers.

Accurate estimates of the total number of neurons located in the wall of the gut are essential for studies of the enteric nervous system (ENS). Though several stains and antibodies are used routinely as pan-neuronal markers, controversies of relative sensitivity and completeness have been difficult to resolve, at least in part because comparisons often must be made across experiments and laboratories. Therefore, we evaluated the efficacy of four putative pan-neuronal markers for the ENS, under comparable conditions. Neurons in the myenteric plexus of wholemounts taken from the small intestines of Fischer 344 rats were stained using Cuprolinic Blue, anti-HuC/D, anti-protein gene product 9.5, or FluoroGold injections followed by permanent labeling with an antibody to the FluoroGold molecule. All four markers had useful features, but both protein gene product 9.5 and FluoroGold were found to be problematic for obtaining reliable counts. As a result, only neurons labeled with either Cuprolinic Blue or anti-HuC/D were compared quantitatively. Based on counts from permanently labeled tissue, Cuprolinic Blue and HuC/D were similarly effective in labeling all neurons. Because the two protocols have different strengths and weaknesses, Cuprolinic Blue and HuC/D provide a complementary set of labels to study the total neuronal population of the ENS.

Gastric satiation is volumetric, intestinal satiation is nutritive.

Gerry Smith's thoughtful survey in his book Satiation (1998) outlined the established principles of gastric and intestinal satiation and delineated several questions still requiring clarification. Experiments since the time of the review have addressed some of these questions. A synthesis of the principles outlined in the Gerry Smith survey and the subsequent experimental results indicates that the direct controls, or neural feedback signals from the GI tract, that limit meal size consist of gastric volumetric signals and intestinal nutritive signals. The two types of negative feedback synergize in the control of feeding, and both are carried by vagal afferents.