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The Importance of Visceral Afferent Fibers

Date: Thursday, December 12th, 2002

Title: “The Role of Visceral Afferent Fibers in Feeding and Malaise”

Speaker: Charles C. Horn, PhD, Monell Chemical Senses Center, Philadelphia, PA

The afferent fibers of the vagus and spanchnic nerves innervating the gastrointestinal (GI) tract and liver sample the quality and quantity of ingested food. Afferent pathways of the vagus are implicated in several important disease processes, including: 1) Responses to food-borne pathogens and toxins- vomiting, nausea, immune-signaling; 2) Gastrointestinal diseases- gastroesophageal reflux, diabetic gastroparesis, functional dyspepsia; 3) Regulation of energy balance- obesity, weight control, diabetes. The sub-diaphragmatic vagus innervates the stomach, intestine, pancreas, liver and portal vein.

Nutritional stimuli, such as fats, carbohydrates and amino acids, as well as osmotic stimuli, such as sodium and water, toxins, temperature, and mechanical (stretch/tension) affect the activity of vagal afferent fibers (1; 2; 9; 12; 17; 19; 25). Vagal afferent fibers are also sensitive to a large number signaling factors: serotonin (5-HT), cholecystokinin (CCK), glucagon-like-peptide-1 (GLP-1), leptin, ghrelin, bombesin, gastrin, histamine, glucagon, insulin, interleukin 1b, neurotensin, somatostatin, adenosine, ATP, histamine, prostaglandin and bradykinin (3; 5; 11; 15; 16; 18; 20; 21; 24). There are three classes of afferent vagal fibers: mucosal endings, intraganglionic endings, and intramuscular arrays. Mucosal endings are free nerve endings that are anatomically close to the mucosal layer of the GI tract. Enteroendocrine cells of the mucosal layer release signaling factors, such as 5-HT and CCK, when stimulated by nutrients and toxins. These signaling factors then act in a paracrine fashion to stimulate afferent fibers of the vagus.

I use a collection of techniques to record the activity of common hepatic branch (CHB) vagal afferent fibers of the rat (13). The CHB of the vagus primarily innervates the upper intestine but also contributes to innervation of the liver, portal vein, stomach and pancreas (4; 22). Rats were anesthetized and paralyzed with Brevital and pancuronium bromide and were subsequently maintained on artificial respiration. Heart rate, blood pressure, body temperature and expired CO2 were monitored to insure the physiological stability of the preparation. Jugular and portal vein catheters were also installed to test the effects of various chemical and nutrient preparations on vagal afferent activity. The electrode array consisted of 8 platinum wires embedded in a silicon platform that was positioned close to the esophagus, below the diaphragm. Each of the four recording electrodes had a separate reference electrode. The ventral trunk of the vagus was cut rostral to the origin of the CHB, and pinned to the recording platform. To eliminate neural signaling from other vagal branches, ventral gastric and accessory celiac branches of the vagus were also transected. Up to four small nerve filaments were teased away from the trunk and wrapped around the recording electrodes. Spike waveforms were extracted and data were processed using principal component analysis, cluster cutting, and inter-spike interval histograms to analyze single unit activity.

Is there integration of input from CCK and 5-HT at the level of GI tract afferent vagal fibers? Hillsley and Grundy showed that mesenteric afferent fibers innervating the jejunum are sensitive to either CCK or 5-HT, but none were sensitive to both neurochemicals (11). However, these recordings did not distinguish between vagal, spinal or intestinofugal mesenteric afferent fibers. The techniques described above were therefore applied to directly record from CHB vagal afferent fibers. Cutting the gastro-duodenal branch of the CHB abolished 80% of its spontaneous activity, indicating that most recorded neuronal activity from the CHB arises from afferent fibers of the GI tract.

To further investigate CHB activity, CCK (100 pM) or 5-HT (10 µg) was infused into the jugular vein. Of 42 single units, 37% responded exclusively to 5-HT, 12% responded exclusively to CCK, 24% responded to both 5-HT and CCK, and 29% responded to neither. This demonstrates that single afferent fibers of the vagus do receive input from both 5-HT and CCK, and the majority of CHB fibers are sensitive to 5-HT.

Daughters et al. (6) demonstrated that CCK and 5-HT interact to inhibit feeding in rats that were re-fed after a 24-hr fast. They found that Ondansetron, a 5-HT3-receptor antagonist, reduced the potency of CCK-induced intake-suppression by 50%. My experiments examined the role of 5-HT3 receptors in ad libitum feeding in the rat. Blockade of 5-HT3 receptors (via intraperitoneal injection of the 5-HT3 receptor antagonist Y-25130) had no significant effect on feeding during either light or dark phases of the 24-hr cycle.

In contrast to the uncertain role of vagal 5-HT3 receptors under natural feeding conditions there is substantial evidence that vagal 5-HT3 receptors are involved in both vomiting and malaise. Some toxins produce vomiting and nausea by stimulating release of 5-HT from enteroendocrine cells, which activates vagal afferent fibers containing 5-HT3 receptors. For example, 5-HT3 receptor antagonists significantly reduce the vomiting and nausea caused by chemotherapy in cancer patients. Intravenous infusion of the chemotherapy agent cisplatin (10 mg/kg) increased CHB vagal activity, which was blocked by infusion of the 5-HT3 antagonist (Y-25130). Cisplatin-induced CHB vagal-activation was also blocked by cutting the gastro-duodenal branch of the vagus indicating that cisplatin affects vagal activity by acting on the GI tract. Rats treated with cisplatin engage in pica, an abnormal feeding activity characterized by consumption of non-nutritive substances such as clay or dirt. This behavior typically occurs after animals have been treated with a toxin and may represent an adaptive response to dilute a toxin’s effect in the body. Cisplatin produced no short-term effect (1-2 hr) on food intake or clay consumption (pica) but did decrease food intake and increase clay consumption by 24 hours. Saeki et al. showed that cisplatin-induced pica was blocked by treatment with a 5-HT3 antagonist (Ondansetron) (23). 5-HT3 receptors are also involved in the development of conditioned taste aversions. Terry-Nathan et al. showed that a 5-HT3 receptor antagonist (tropisetron) blocked the conditioned taste aversion to an imbalanced amino acid diet in rats (26). Research has indicated that the CHB of the vagus is important for the detection of amino acids (7). The 5-HT3 receptor antagonist Ondansetron significantly increased enjoyment derived from eating among cancer patients who were suffering from anorexia-cachexia syndrome, which is characterized by decreased protein metabolism (8). Despite this effect, the treatment failed to influence weight loss in these patients.

Vagal afferent fibers containing 5-HT3 receptors may also play a role in intestinal carbohydrate detection. Zhu et al. recorded nodose ganglia neurons of the rat that were sensitive to intestinal infusion of glucose and maltose. The response of vagal neurons to carbohydrate was blocked by treatment with the 5-HT3 receptor antagonist Granisetron (27). Furthermore, Kim et al. showed that release of 5-HT from human enteroendocrine cells could by induced by glucose, using an in vitro preparation (14). Vagal and cell culture data suggest that enteroendocrine cells detect glucose concentration in the intestine and release 5-HT, which activates vagal afferent fibers that contain 5-HT3 receptors.

Is there a role for peripheral CCK in nausea and malaise? Given the strong evidence for involvement of a 5-HT3 vagal pathway in vomiting and malaise, part of the feeding-inhibitory effect produced by CCK treatment may result from producing malaise (6). Fried and Feinle showed that CCK participates in the pain, nausea and discomfort experienced during duodenal lipid infusions among patients with functional dyspepsia (10). Functional dyspeptic patients who were given duodenal lipid infusions reported lower levels of fullness, discomfort, nausea, pain and bloating if they had also received an injection of the CCK-A receptor antagonist, Dexloxiglumide.

In summary, afferent fibers of the vagus are involved in the detection of nutrients and toxins at pre- and post-absorptive sites, which leads to appropriate behavioral responses. CCK and 5-HT are both released from enteroendocrine cells in the GI tract in response to ingested chemicals, and act in a paracrine fashion to stimulate vagal afferent fibers. Although there is the potential for some functional overlap, CCK in the GI tract appears to be involved in the feedback control of ingestion, while 5-HT (5-HT3) in the GI tract is involved in emesis/malaise-associated behaviors.


Q: At what level of the vagus did you find bilateral afferentation?
A: At the cervical level. I recorded the compound action potential from both left and right cervical vagi while electrically stimulating the ventral and dorsal subdiaphragmatic vagal trunks. I was surprised to find evidence of crossing of vagal fibers in the thorax. For example, I was able to record a compound action potential from the right cervical vagus when I stimulated the ventral or dorsal subdiaphragmatic vagal trunks. Anatomical evidence for thoracic crossing of vagal fibers in the rat is lacking.

Q: What’s the plasticity of the vagus?
A: The visceral afferent fibers of the GI tract appear to re-organize following lesions of the vagus. For example, Grundy has shown that cisplatin evokes a 5-HT3 mediated response in mesenteric afferent fibers and following vagotomy cisplatin evokes a 5-HT3-independent mesenteric afferent response. This suggests that vagotomy has produced a change in how intestino-fugal or spinal afferents respond to toxins.

Q: Have you compared results obtained by principle component analysis with those obtained by more conventional means?
A: Yes, I have compared principal component analysis with template matching. The results are similar. I think the critical issue is to use an analysis procedure that takes into account the whole waveform, e.g., principal component analysis or template-matching, so that small not so obvious features can be used to distinguish single units in a multi-unit recording.

Q: What types of spikes are likely to be missed, and how frequently, if data are not analyzed with principle components?
A: Spikes that have a low signal-to-noise ratio will not be included. I typically get a 4:1 signal-to noise-ratio and this is adequate for single unit selection using principal component analysis. Spikes that are close to the baseline noise cannot be differentiated into single units. If your electrophysiology technique is good, i.e. grounding, etc., signal-to-noise ratios should not be a problem. The more critical issue is selecting and grouping spikes that should not be grouped together as single units. An analysis of the refractory, using inter-spike interval histograms, is a good way to determine if you have single unit activity.

Q: Are the 5-HT fibers mostly C-type fibers?
A: Yes, most are C-fibers.

Q: Why did you study responses in 5-HT/CCK sensing neurons in the post-prandial state rather than the fasted state?
A: Animals are studied during the day for neurophysiology experiments. During this time there is little food intake in the rat. Fasting significantly alters metabolic variables. Most of my behavioral studies are in ad libitum fed animals and I therefore use ad libitum fed rats for neurophysiology experiments to provide a more similar comparison between conditions.

Q: Does 5-HT reduce food intake when it is given peripherally?
A: When we gave the 5-HT3 agonist peripherally, it reduced food intake.

Q: Is the 5-HT3 receptor-antagonist the only receptor-antagonist that blocks nausea?
A: No. In the study by Fried and Feinle the CCK-A receptor antagonist, dexloxiglumide, was effective for reducing nausea in patients with functional dyspepsia during intestinal lipid infusion.

Q: How do 5-HT antagonists affect feeding, if at all?
A: Thus far we’ve seen no effect of 5-HT3 antagonists on ad libitum feeding.


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