Eating in mice with gastric bypass surgery causes exaggerated activation of brainstem anorexia circuit.

BACKGROUND/OBJECTIVE: Obesity and metabolic diseases are at an alarming level globally and increasingly affect children and adolescents. Gastric bypass and other bariatric surgeries have proven remarkably successful and are increasingly performed worldwide. Reduced desire to eat and changes in eating behavior and food choice account for most of the initial weight loss and diabetes remission after surgery, but the underlying mechanisms of altered gut-brain communication are unknown. SUBJECTS/METHODS: To explore the potential involvement of a powerful brainstem anorexia pathway centered around the lateral parabrachial nucleus (lPBN), we measured meal-induced neuronal activation by means of c-Fos immunohistochemistry in a new high-fat diet-induced obese mouse model of Roux-en-Y gastric bypass (RYGB) at 10 and 40 days after RYGB or sham surgery. RESULTS: Voluntary ingestion of a meal 10 days after RYGB, but not after sham surgery, strongly and selectively activates calcitonin gene-related peptide neurons in the external lPBN as well as neurons in the nucleus tractus solitarius, area postrema and medial amygdala. At 40 days after surgery, meal-induced activation in all these areas was greatly diminished and did not reach statistical significance. CONCLUSIONS: The neural activation pattern and dynamics suggest a role of the brainstem anorexia pathway in the early effects of RYGB on meal size and food intake that may lead to adaptive neural and behavioral changes involved in the control of food intake and body weight at a lower level. However, selective inhibition of this pathway will be required for a more causal implication.

Leptin deficient ob/ob mice and diet-induced obese mice responded differently to Roux-en-Y bypass surgery.

OBJECTIVE: Weight regain contributes to the therapeutic failure in 15-20% of type 2 diabetic patients after Roux-en-Y gastric bypass surgery (RYGB), and the mechanism remains largely unknown. This study was conducted to explore the mechanism of weight regain. RESEARCH DESIGN: Wild-type (WT) diet-induced obese (DIO) mice were used to mimic human obesity, and ob/ob mice were used for leptin deficiency-induced obesity. Two groups of mice were compared in weight regain for 10 months after RYGB. Weight loss, food intake, fecal energy loss and energy expenditure were monitored in the study of weight regain. Fasting insulin, insulin tolerance and homeostatic model assessment-insulin resistance were tested for insulin sensitivity under the weight regain. Weight loss from RYGB and calorie restriction was compared for the impact in insulin sensitivity. RESULTS: In WT mice, RYGB induced a sustained weight loss and insulin sensitization over the sham operation in this 10-month study. However, RYGB failed to generate the same effects in leptin-deficient ob/ob mice, which suffered a weight regain over the pre-surgery level. In ob/ob mice, body weight was reduced by RYGB transiently in the first week, recovered in the second week and increased over the baseline thereafter. Weight loss was induced by RYGB relative to that of sham mice, but the loss was not sufficient to keep body weight below the pre-surgery levels. In addition, insulin sensitivity was not improved by the weight loss. The response to RYGB was improved in ob/ob mice by 2 weeks of leptin treatment. Weight loss from calorie restriction had an equivalent effect on insulin sensitization compared with that of RYGB. CONCLUSION: Those data demonstrate that ob/ob mice and DIO mice responded differently to RYGB surgery, suggesting that leptin may be one of the factors required for RYGB to prevent weight regain and diabetes recurrence.

Food reward functions as affected by obesity and bariatric surgery.

Roux-en-Y gastric bypass surgery (RYGB) remains to be the most effective long-term treatment for obesity and its associated comorbidities, but the specific mechanisms involved remain elusive. Because RYGB patients appear to no longer be preoccupied with thoughts about food and are satisfied with much smaller meals and calorically dilute foods, brain reward mechanisms could be involved. Just as obesity can produce maladaptive alterations in reward functions, reversal of obesity by RYGB could normalize these changes or even further reset the food reward system through changes in gut hormone secretion, aversive conditioning and/or secondary effects of weight loss. Future studies with longitudinal assessments of reward behaviors and their underlying neural circuits before and after surgery will be necessary to uncover the specific mechanisms involved. Such new insights could be the base for future 'knifeless' pharmacological and behavioral approaches to obesity.

Roux-en-Y gastric bypass surgery changes food reward in rats.

CONTEXT: Roux-en-Y gastric bypass surgery (RYGB) is currently the most effective treatment for morbid obesity, and clinical studies suggest that RYGB patients change food preferences and the desire to eat. OBJECTIVE: To examine hedonic reactions to palatable foods and food choice behavior in an established rat model of RYGB. METHODS AND DESIGN: Male Sprague-Dawley (SD) rats and selected line obesity-prone rats that were rendered obese on a high-fat diet underwent RYGB or sham surgery and were tested for 'liking' and 'wanting' of palatable foods at different caloric densities 4-6 months after surgery. RESULTS: Compared with sham-operated (obese) and age-matched lean control rats, RYGB rats of both models exhibited more positive orofacial responses to low concentrations of sucrose but fewer to high concentrations. These changes in 'liking' by RYGB rats were translated into a shift of the concentration-response curve in the brief access test, with more vigorous licking of low concentrations of sucrose and corn oil, but less licking of the highest concentrations. The changes in hedonic evaluation also resulted in lower long-term preference/acceptance of high-fat diets compared with sham-operated (obese) rats. Furthermore, the reduced 'wanting' of a palatable reward in the incentive runway seen in sham-operated obese SD rats was fully restored after RYGB to the level found in lean control rats. CONCLUSIONS: The results suggest that RYGB leads to a shift in hedonic evaluation, favoring low over high calorie foods and restores obesity-induced alterations in 'liking' and 'wanting'. It remains to be determined whether these effects are simply due to weight loss or specific changes in gut-brain communication. Given the emerging evidence for modulation of cortico-limbic brain structures involved in reward mechanisms by gut hormones, RYGB-induced changes in the secretion of these hormones could potentially be mediating these effects.

Chronic suppression of µ-opioid receptor signaling in the nucleus accumbens attenuates development of diet-induced obesity in rats.

OBJECTIVE: To test the hypothesis that micro-opioid receptor signaling in the nucleus accumbens contributes to hedonic (over)eating and obesity. To investigate the effects of chronic micro-opioid antagonism in the nucleus accumbens core or shell on intake of a palatable diet, and the development of diet-induced obesity in rats. METHODS AND DESIGN: Chronic blockade of micro-opioid receptor signaling in the nucleus accumbens core or shell was achieved by means of repeated injections (every 4-5 days) of the irreversible receptor antagonist beta-funaltrexamine (BFNA) over 3-5 weeks. The diet consisted of either a choice of high-fat chow, chocolate-flavored Ensure and regular chow (each nutritionally complete) or regular chow only. Intake of each food item, body weight and body fat mass were monitored throughout the study. RESULTS: The BFNA injections aimed at either the core or shell of the nucleus accumbens resulted in significantly attenuated intake of palatable diet, body weight gain and fat accretion, compared with vehicle control injections. The injection of BFNA in the core did not significantly change these parameters in chow-fed control rats. The injection of BFNA in the core and shell differentially affected intake of the two palatable food items: in the core, BFNA significantly reduced the intake of high-fat, but not of Ensure, whereas in the shell, it significantly reduced the intake of Ensure, but not of high-fat, compared with vehicle treatment. CONCLUSIONS: Endogenous micro-opioid receptor signaling in the nucleus accumbens core and shell is necessary for palatable diet-induced hyperphagia and obesity to fully develop in rats. Sweet and non-sweet fatty foods may be differentially processed in subcomponents of the ventral striatum.

Appetite control and energy balance regulation in the modern world: reward-driven brain overrides repletion signals.

Powerful biological mechanisms evolved to defend adequate nutrient supply and optimal levels of body weight/adiposity. Low levels of leptin indicating food deprivation and depleted fat stores have been identified as the strongest signals to induce adaptive biological actions such as increased energy intake and reduced energy expenditure. In concert with other signals from the gut and metabolically active tissues, low leptin levels trigger powerful activation of multiple peripheral and brain systems to restore energy balance. It is not just neurons in the arcuate nucleus, but many other brain systems involved in finding potential food sources, smelling and tasting food, and learning to maximize rewarding effects of foods, that are affected by low leptin. Food restriction and fat depletion thus lead to a 'hungry' brain, preoccupied with food. By contrast, because of less (adaptive thrifty fuel efficiency) or lost (lack of predators) evolutionary pressure, the upper limits of body weight/adiposity are not as strongly defended by high levels of leptin and other signals. The modern environment is characterized by the increased availability of large amounts of energy-dense foods and increased presence of powerful food cues, together with minimal physical procurement costs and a sedentary lifestyle. Much of these environmental influences affect cortico-limbic brain areas concerned with learning and memory, reward, mood and emotion. Common obesity results when individual predisposition to deal with a restrictive environment, as engraved by genetics, epigenetics and/or early life experience, is confronted with an environment of plenty. Therefore, increased adiposity in prone individuals should be seen as a normal physiological response to a changed environment, not in the pathology of the regulatory system. The first line of defense should ideally lie in modifications to the environment and lifestyle. However, as such modifications will be slow and incomplete, it is equally important to gain better insight into how the brain deals with environmental stimuli and to develop behavioral strategies to better cope with them. Clearly, alternative therapeutic strategies such as drugs and bariatric surgery should also be considered to prevent or treat this debilitating disease. It will be crucial to understand the functional crosstalk between neural systems responding to metabolic and environmental stimuli, i.e. crosstalk between hypothalamic and cortico-limbic circuitry.

EmsB, a tandem repeated multi-loci microsatellite, new tool to investigate the genetic diversity of Echinococcus multilocularis.

In order to explore the genetic diversity within Echinococcus multilocularis (E. multilocularis), the cestode responsible for the alveolar echinococcosis (AE) in humans, a microsatellite, composed of (CA) and (GA) repeats and designated EmsB, was isolated and characterized in view of its nature and potential field application. PCR-amplification with specific primers exhibited a high degree of size polymorphism between E. multilocularis and Echinococcus granulosus sheep (G1) and camel (G6) strains. Fluorescent-PCR was subsequently performed on a panel of E. multilocularis isolates to assess intra-species polymorphism level. EmsB provided a multi-peak profile, characterized by tandemly repeated microsatellite sequences in the E. multilocularis genome. This "repetition of repeats" feature provided to EmsB a high discriminatory power in that eight clusters, supported by bootstrap p-values larger than 95%, could be defined among the tested E. multilocularis samples. We were able to differentiate not only the Alaskan from the European samples, but also to detect different European isolate clusters. In total, 25 genotypes were defined within 37 E. multilocularis samples. Despite its complexity, this tandem repeated multi-loci microsatellite possesses the three important features for a molecular marker, i.e. sensitivity, repetitiveness and discriminatory power. It will permit assessing the genetic polymorphism of E. multilocularis and to investigate its spatial distribution in detail.

Does gastric bypass surgery change body weight set point?

The relatively stable body weight during adulthood is attributed to a homeostatic regulatory mechanism residing in the brain which uses feedback from the body to control energy intake and expenditure. This mechanism guarantees that if perturbed up or down by design, body weight will return to pre-perturbation levels, defined as the defended level or set point. The fact that weight re-gain is common after dieting suggests that obese subjects defend a higher level of body weight. Thus, the set point for body weight is flexible and likely determined by the complex interaction of genetic, epigenetic and environmental factors. Unlike dieting, bariatric surgery does a much better job in producing sustained suppression of food intake and body weight, and an intensive search for the underlying mechanisms has started. Although one explanation for this lasting effect of particularly Roux-en-Y gastric bypass surgery (RYGB) is simple physical restriction due to the invasive surgery, a more exciting explanation is that the surgery physiologically reprograms the body weight defense mechanism. In this non-systematic review, we present behavioral evidence from our own and other studies that defended body weight is lowered after RYGB and sleeve gastrectomy. After these surgeries, rodents return to their preferred lower body weight if over- or underfed for a period of time, and the ability to drastically increase food intake during the anabolic phase strongly argues against the physical restriction hypothesis. However, the underlying mechanisms remain obscure. Although the mechanism involves central leptin and melanocortin signaling pathways, other peripheral signals such as gut hormones and their neural effector pathways likely contribute. Future research using both targeted and non-targeted 'omics' techniques in both humans and rodents as well as modern, genetically targeted, neuronal manipulation techniques in rodents will be necessary.

Melanin concentrating hormone innervation of caudal brainstem areas involved in gastrointestinal functions and energy balance.

Neural signaling by melanin-concentrating hormone and its receptor (SLC-1) has been implicated in the control of energy balance, but due to the wide distribution of melanin-concentrating hormone-containing fibers throughout the neuraxis, its critical sites of action for a particular effect have not been identified. The present study aimed to anatomically and functionally characterize melanin-concentrating hormone innervation of the rat caudal brainstem, as this brain area plays an important role in the neural control of ingestive behavior and autonomic outflow. Using retrograde tracing we demonstrate that a significant proportion (5-15%) of primarily perifornical and far-lateral hypothalamic melanin-concentrating hormone neurons projects to the dorsal vagal complex. In the caudal brainstem, melanin-concentrating hormone-ir axon profiles are distributed densely in most areas including the nucleus of the solitary tract, dorsal motor nucleus of the vagus, and sympathetic premotor areas in the ventral medulla. Close anatomical appositions can be demonstrated between melanin-concentrating hormone-ir axon profiles and tyrosine hydroxylase, GABA, GLP-1, NOS-expressing, and nucleus of the solitary tract neurons activated by gastric nutrient infusion. In medulla slice preparations, bath application of melanin-concentrating hormone inhibited in a concentration-dependent manner the amplitude of excitatory postsynaptic currents evoked by solitary tract stimulation via a pre-synaptic mechanism. Fourth ventricular administration of melanin-concentrating hormone (10 microg) in freely moving rats decreased core body temperature but did not change locomotor activity and food and water intake. We conclude that the rich hypothalamo-medullary melanin-concentrating hormone projections in the rat are mainly inhibitory to nucleus of the solitary tract neurons, but are not involved in the control of food intake. Projections to ventral medullary sites may play a role in the inhibitory effect of melanin-concentrating hormone on energy expenditure.

Appetite and body weight regulation after bariatric surgery.

Bariatric surgery continues to be remarkably efficient in treating obesity and type 2 diabetes mellitus and a debate has started whether it should remain the last resort only or also be used for the prevention of metabolic diseases. Intense research efforts in humans and rodent models are underway to identify the critical mechanisms underlying the beneficial effects with a view towards non-surgical treatment options. This non-systematic review summarizes and interprets some of this literature, with an emphasis on changes in the controls of appetite. Contrary to earlier views, surgery-induced reduction of energy intake and subsequent weight loss appear to be the main drivers for rapid improvements of glycaemic control. The mechanisms responsible for suppression of appetite, particularly in the face of the large weight loss, are not well understood. Although a number of changes in food choice, taste functions, hedonic evaluation, motivation and self-control have been documented in both humans and rodents after surgery, their importance and relative contribution to diminished appetite has not yet been demonstrated. Furthermore, none of the major candidate mechanisms postulated in mediating surgery-induced changes from the gut and other organs to the brain, such as gut hormones and sensory neuronal pathways, have been confirmed yet. Future research efforts should focus on interventional rather than descriptive approaches in both humans and rodent models.

CNS modulation of pancreatic endocrine function : Multiple modes of expression.

The involvement of the CNS in pancreatic hormone release has been studied. 1.) It has been shown that one source of vagal efferent fibers capable of facilitating insulin secretion originated in the rostral half of the nucleus ambiguus. 2.) Acute lesions of the ventromedial hypothalamus resulted in hyperinsulinaemia that could be abolished by acute vagotomy. 3.) Chronic lesions of the ventromedial hypothalamus increased secretion of insulin and glucagon, and decreased secretion of somatostatin when the pancreas was subsequently isolated and perfused. These changes were attributed to altered cholinergic activity related to previous ventromedial hypothalamic lesions as they could be reversed toward normal by atropine infusion or mimicked by the cholinergic agonist, methacholine. 4.) Electrical stimulation of the lateral hypothalamus in anaesthetized rats produced both an inhibitory component of insulin secretion, probably related to adrenergic stimulation, and a stimulatory component, probably due to the release into the blood of factor(s) that promote insulin secretion. 5.) The anatomical organization of brain of the genetically obese (ob/ob) mice is abnormal. These abnormalities could be involved in the endocrinological disturbances of these animals.

Cephalic phase, reflex insulin secretion neuroanatomical and physiological characterization.

Using chronically catheterized, freely moving male Wistar rats, we have shown that the sweet taste of a saccharin solution reliably triggers a rapid cephalic phase insulin response (CPIR), in the absence of any significant change of glycemia. To establish the neural mediation of this reflex response we used rats that were cured from streptozotocin diabetes by intrahepatic islet-transplantation as a denervated B-cell preparation. The complete lack of any saccharin-induced CPIR in these rats suggests that it is indeed mediated by the peripheral autonomic nervous system, and that the insulin-stimulating gastrointestinal hormones are not involved in this response. It was further found that this reflex insulin secretion is not easily extinguishable and thus might have an unconditioned component. To investigate the central neural pathways involved in this reflex response we used both electrophysiological methods in anesthetized and semi-micro CNS manipulations in freely moving rats. On the basis of our preliminary results, and several reports, using the decerebrate rat preparation for measuring behavioral or saliva secretory oral taste reactivity, it appears that CPIR might be organized at the brain stem/midbrain level, receiving strong modulatory influences from the diencephalon. But much further work has to be done to establish the central nervous circuitry. Finally, in two experiments, aiming at the question of how important and physiologically relevant the CPIR might be, we found that, on one hand, its lack can result in pathological oral glucose tolerance and on the other hand its exaggeration might contribute to the behavioral reaction to highly palatable sweet food and the resulting development of dietary obesity.

Acute hyperinsulinemia and its reversal by vagotomy after lesions of the ventromedial hypothalamus in anesthetized rats.

The acute effect of bilateral electrolytic ventromedial hypothalamic lesions (20-25-m Coulomb stainless steel electrodes) on plasma levels of insulin and glucose was studied in anesthetized rats to determine early effects that would occur before hyperphagia and obesity. In rats fed ad libitum, lesions in the ventromedial hypothalamus (VMH) but not in the cortex produced a marked increase in circulating insulin levels (starting at 20 min postlesion) and a small increase in glycemia which, however, was not significant and could therefore not be the cause of increased insulin secretion. Hyperinsulinemia after VMH lesions was more pronounced when glucose was infused iv at a rate of 7-8 mg/kg . min. Bilateral subdiaphragmatic vagotomy, performed 50 min after VMH lesions, immediately and completely reversed the observed hyperinsulinemia. With the exception of a tendency of lesions producing the highest degree of hyperinsulinemia to be slightly larger than the lesions not producing any hyperinsulinemia, no statement about the critical involvement of a specific hypothalamic locus can be made. It is concluded that electrolytic VMH destruction causes immediate hypersecretion of the pancreatic B cell, an effect that requires the integrity of the vagus nerves. Further localization of the central circuitry responsible for this mechanism, however, will require more specific methods than electrolytic lesions.

Increases in plasma insulin levels in response to electrical stimulation of the dorsal motor nucleus of the vagus nerve.

In order to investigate the physiological counterpart of the anatomical finding showing that the dorsal motor nucleus of the vagus nerve (DMX) is a source of efferent vagal fibers innervating the pancreas, unilateral electrical stimulation using monopolar electrodes (50 microA, 30 Hz, 0.2 msec) at a glycemia of 150 mg/100 ml was performed in normal anesthetized rats. DMX stimulation resulted in rapid (within 1 min) rise in plasma insulin levels (greater than or equal to 200%). Stimulation of the nucleus of tractus solitarius, anatomically connected to DMX, also produced a 50% increase in insulinemia. The effect of DMX stimulation was almost completely abolished by atropine pretreatment or acute bilateral subdiaphragmatic vagotomy. The effect of DMX stimulation was not potentiated by the alpha-adrenergic blocker (infusion of phentolamine) indicating that no inhibitory fiber was recruited during DMX stimulation. It is concluded that DMX is connected to the endocrine pancreas exclusively via vagal fibers and has a role in neurally mediated insulin release.

Brain stem infusion of the gamma-aminobutyric acid antagonist bicuculline increases plasma insulin levels in the rat.

Previous neuroanatomical and physiological studies have indicated that nucleus ambiguus (Amb) is one source of vagal motoneurons in the brain stem that innervates the pancreas and which, when stimulated, increases insulin release. To investigate one of the neurotransmitter inputs to Amb neurons and its relation to insulin secretion, bicuculline, a specific gamma-aminobutyric acid (GABA) antagonist, was infused bilaterally into the Amb region as well as into a neighboring area, the rostral level of the lateral nucleus tractus solitarius (nts) of anesthetized male rats. Experiments were carried out in the presence or absence of the alpha-adrenergic blocker phentolamine. In the absence of phentolamine, no increases in plasma insulin levels were seen after bicuculline or vehicle infusion into the Amb region or after bicuculline infusion into the nts, while plasma glucose levels were significantly increased. In the presence of phentolamine, bicuculline infusion into the Amb region led to a prompt and significant increase in plasma insulin levels that could not be accounted for by changes in glycemia. The infusion of vehicle into Amb or of bicuculline into nts produced small or insignificant increases in plasma insulin levels. These results suggest that Amb neurons capable of modulating plasma insulin levels are under tonic GABA inhibition, an effect that appears to be specific for Amb neurons, since bicuculline infusion into another brain stem nucleus (nts) had no effect on insulin release. The fact that phentolamine pretreatment was necessary to reveal the bicuculline-induced effects corroborates previous studies showing that in addition to a central nervous system inhibition of vagal motoneurons by GABA, there is a tonic sympathetic inhibitory input to the endocrine pancreas capable of masking any disinhibition of vagal motoneurons. The physiological role of GABA inhibition of Amb neurons that innervate the pancreatic beta-cells remains to be determined.

Intraportal islet transplantation: functional assessment in conscious unrestrained rats.

We have found that rats with transplanted pancreatic islets, when compared to normal rats, have a delayed onset of insulin release in response to orally, but not to iv administered glucose. Furthermore, while glucose tolerance of the rats with transplanted islets was similar to that of normal controls when the glucose was administered iv, the tolerance was markedly less when it was administered orally. These tests were carried out using permanently implanted cardiac catheters and chronic oral fistulae and were conducted at a time when the body weight of the transplanted animals had returned to levels similar to those of the controls. During the tests the rats were conscious and unrestrained. The difference in the fine control of insulin secretion in transplanted islets from that in the normal pancreas may be due to defective innervation of such islets. These results may have implications for the use of transplanted islets in the control of diabetes mellitus in man. The methods employed can be further used to define other areas in which the response of transplanted islets in rats differs from that of the normal pancreas.

Anatomical demonstration of vagal input to nicotinamide acetamide dinucleotide phosphate diaphorase-positive (nitrergic) neurons in rat fundic stomach.

Recent pharmacological evidence suggests that the nonadrenergic, noncholinergic (NANC) vagal inhibitory input responsible for receptive relaxation of the fundic stomach is mediated by nitric oxide-synthesizing enteric neurons. To demonstrate anatomically such direct vagal inputs to neurochemically identified enteric neurons, we utilized the nicotinamide acetamide dinucleotide phosphate (NADPH)-diaphorase histochemical reaction in conjunction with selective anterograde labeling of vagal efferents or afferents. Approximately 30% of all myenteric neurons of the fundic myenteric plexus stained positive for NADPH diaphorase, and the principal recipient of axonal projections from NADPH diaphorase-positive neurons was the circular muscle layer. In a group of animals showing the most complete labeling of vagal efferent preganglionics with the carbocyanine dye DiA, quantitative analysis of the half of the ventral fundic wall closer to the greater curvature revealed that 46.8% +/- 4.4% of all myenteric neurons received some degree of vagal contacts and that 30.5% +/- 6.6% of such vagally contacted neurons were also NADPH diaphorase positive. In another group of rats with the most successful selective labeling of vagal afferents through DiI injections into the left nodose ganglion, analysis of select ganglia throughout the ventral fundic wall revealed that, of a total of 454 neurons with vagal afferent contacts, 34.8% +/- 2.8% were NADPH diaphorase positive. These findings support the view that, in the fundic stomach, some vagal preganglionic efferents terminate on nitric oxide-synthesizing neurons that, in turn, project to and relax the external smooth muscle layers. Furthermore, vagal afferent endings also contact NADPH diaphorase-positive neurons, suggesting the possibility of local axon reflexes originating from smooth muscular in-series tension receptors and terminating on nitrergic neurons of the myenteric plexus.

Morphological analysis of vagal input to gastrin releasing peptide and vasoactive intestinal peptide containing neurons in the rat glandular stomach.

Vagal preganglionic efferents to the rat stomach were labeled anterogradely by injecting the fluorescent carbocyanine dye DiA into the dorsal motor nucleus in vivo. Enteric neurons were labeled in toto by intraperitioneal administration of Fluorogold, and neurochemically characterized by simultaneous single- and double-label immunocytochemistry. Single peptide immunocytochemistry revealed that in all three major areas of the stomach, about one-third of all gastrin-releasing peptide immunoreactive (GRP-IR) neurons in the myenteric plexus, received vagal contacts. Because the proportion of GRP-IR neurons was 32% in the fundus, 23% in the corpus, and only 8% in the antrum, the absolute number of vagally contacted GRP-IR neurons per cm2 was also different. Double-label immunocytochemistry revealed colocalization of vasoactive intestinal peptide immunoreactivity (VIP-IR) in 45%, and of enkephalin immunoreactivity (ENK-IR) in about 30%, of the GRP-IR myenteric neurons. A subpopulation of myenteric neurons colocalized GRP-IR and VIP-IR and projects almost exclusively to the gastrin cell-rich basal mucosa of the antrum and the oxyntic mucosa of the corpus. Another subpopulation containing GRP-IR, but not VIP-IR, projects mainly to the myenteric plexus itself and the external muscle layers, particularly the longitudinal muscle. A third group of neurons containing VIP-IR but not GRP-IR projects heavily to the circular muscle layer, the muscularis mucosae, and to other myenteric neurons. Vagal input to these three subpopulations seems not to be selective, in that an equal proportion of about 20 to 30% of each group was vagally contacted. Vagal inputs to these neurochemically and topographically distinct enteric neurons provide the basis for the physiological vagal control of gastrin release, gastric acid secretion, and gastric motility.

Vagal afferent innervation of the rat fundic stomach: morphological characterization of the gastric tension receptor.

Although the gastric tension receptor has been characterized behaviorally and electrophysiologically quite well, its location and structure remains elusive. Therefore, the vagal afferents to the rat fundus (forestomach or nonglandular stomach) were anterogradely labeled in vivo with injections of the carbocyanine dye Dil into the nodose ganglia, and the nerves and ganglia of the enteric nervous system were labeled in toto with intraperitoneal Fluorogold injection. Dissected layers and cryostat cross sections of the fundic wall were mounted in glycerin and analyzed by means of conventional and laser scanning confocal microscopy. Particularly in the longitudinal, and to a lesser extent in the circular, smooth muscle layers, Dil-labeled fibers and terminals were abundant. These processes, which originated from fibers coursing through the myenteric ganglia and connectives, entered either muscle coat and then ran parallel to the respective muscle fibers, often for several millimeters. They ran in close association with the Fluorogold-labeled network of interstitial cells of Cajal, upon which they appeared to form multiple spiny appositions or varicosities. In the myenteric plexus, two different types of afferent vagal structures were observed. Up to 300 highly arborizing endings forming dense accumulations of small puncta similar to the esophageal intraganglionic laminar endings (Rodrigo et al., '75 Acta Anat. 92:79-100) were found in the fundic wall ipsilateral to the injected nodose ganglion. They often covered small clusters of myenteric neurons or even single isolated ganglion cells (mean = 5.8 neurons) and tended to extend throughout the neuropil of the ganglia. In a second pattern, fine varicose fibers with less profuse arborizations innervated mainly the central regions of myenteric ganglia. Camera lucida analyses established that single vagal afferent fibers had separate collaterals in both a smooth muscle layer and the myenteric ganglia. Finally, Dil-labeled afferent vagal fibers were also found in the submucosa and mucosa. Control experiments in rats with supranodose vagotomy as well as rats with Dil injections directly in the distal cervical vagus ruled out the possibility of colabeling of afferent fibers of passage. In triple labeling experiments, in conjunction with Dil labeling of afferents and Fluorogold labeling of enteric neurons, the carbocyanine dye DiA was injected into the dorsal motor nucleus of the vagus to anterogradely label the efferent vagal fibers and terminals. The different distributions and morphological characteristics of the vagal afferents and efferents could be simultaneously compared. In some instances the same myenteric ganglion was apparently innervated by an afferent laminar ending and an efferent terminal.(ABSTRACT TRUNCATED AT 400 WORDS)