Hindbrain administration of NMDA receptor antagonist AP-5 increases food intake in the rat

Hindbrain administration of MK-801, a noncompetitive N-methyl-D-aspartate (NMDA) channel blocker, increases meal size, suggesting NMDA receptors in this location participate in control of food intake. However, dizocilpine (MK-801) reportedly antagonizes some non-NMDA ion channels. Therefore, to further assess hindbrain NMDA receptor participation in food intake control, we measured deprivation-induced intakes of 15% sucrose solution or rat chow after intraperitoneal injection of either saline vehicle or D(-)-2-amino-5-phosphonopentanoic acid (AP5), a competitive NMDA receptor antagonist, to the fourth ventricular, or nucleus of the solitary tract (NTS). Intraperitoneal injection of AP5 (0.05, 0.1, 1.0, 3.0, and 5.0 mg/kg) did not alter 30-min sucrose intake at any dose (10.7 +/- 0.4 ml, saline control) (11.0 +/- 0.8, 11.2 +/- 1.0, 11.2 +/- 1.0, 13.1 +/- 2.2, and 11.0 +/- 1.9 ml, AP5 doses, respectively). Fourth ventricular administration of both 0.2 mug (16.7 +/- 0.6 ml) and 0.4 mug (14.9 +/- 0.5 ml) but not 0.1 and 0.6 mug of AP5 significantly increased 60-min sucrose intake compared with saline (11.2 +/- 0.4 ml). Twenty-four hour chow intake also was increased compared with saline (AP5: 31.5 +/- 0.1 g vs. saline: 27.1 +/- 0.6 g). Furthermore, rats did not increase intake of 0.2% saccharin after fourth ventricular AP5 administration (AP5: 9.8 +/- 0.7 ml, vs. saline: 10.5 +/- 0.5 ml). Finally, NTS AP5 (20 ng/30 nl) significantly increased 30- (AP5: 17.2 +/- 0.7 ml vs. saline: 14.6 +/- 1.7 ml), and 60-min (AP5: 19.4 +/- 0.6 ml vs. saline: 15.5 +/- 1.4 ml) sucrose intake, as well as 24-h chow intake (AP5: 31.6 +/- 0.3 g vs. saline: 26.1 +/- 1.2 g). These results support the hypothesis that hindbrain NMDA receptors participate in control of food intake and suggest that this participation also may contribute to control of body weight over a 24-h period.     

NMDA receptor blockade attenuates CCK-induced reduction of real feeding but not sham feeding

Systemic injection of MK-801, a noncompetitive antagonist of N-methyl-D-aspartate (NMDA) receptor ion channels, increases meal size and delays satiation. We examined whether MK-801 increases food intake by directly interfering with actions of cholecystokinin (CCK). Prior administration of MK-801 (100 microg/kg ip) reversed the inhibitory effects of CCK-8 (2 and 4 microg/kg ip) on real feeding of both liquid and solid foods. MK-801 alone did not alter 30-min sham intake of 15% sucrose compared with intake after saline. Furthermore, while CCK-8 (2 or 4 microg/kg ip) reduced sham intake, this reduction was not attenuated by MK-801 pretreatment. To ascertain whether MK-801 attenuation of CCK-induced reduction of real feeding was associated with attenuated inhibition of gastric emptying, we tested the effect of MK-801 pretreatment on CCK-induced inhibition of gastric emptying of 5-ml saline loads. Ten-minute gastric emptying was accelerated after MK-801 (3.9 +/- 0.2 ml) compared with saline vehicle (2.72 +/- 0.2 ml). CCK-8 (0.5 microg/kg ip) reduced 10-min emptying to 1.36 +/- 0.3 ml. Pretreatment with MK-801 did not significantly attenuate CCK-8-induced reduction of gastric emptying (0.9 +/- 0.4 ml). This series of experiments demonstrates that blockade of NMDA ion channels reverses inhibition of real feeding by CCK. However, neither inhibition of sham feeding nor inhibition of gastric emptying by CCK is attenuated by MK-801. Therefore, increased food intake after NMDA receptor blockade is not caused by a direct interference with CCK-induced satiation. Rather, increased real feeding, either in the presence or absence of CCK, depends on blockade of NMDA receptor participation in other post-oral feedback signals such as gastric sensation or gastric tone.     

Cholinergic neurotransmission participates in increased food intake induced by NMDA receptor blockade

MK-801, a noncompetitive N-methyl-d-aspartate (NMDA) receptor antagonist, enhances gastric emptying while increasing food intake. Although our previously reported results implicate the vagus in MK-801's effect on feeding, it is not clear whether vagal motor fibers participate in the feeding response. Control of gastric emptying is exerted, in part, by cholinergic vagal motor neurons. Therefore, we examined the ability of MK-801 to increase meal size in the presence or absence of the muscarinic receptor antagonist atropine methyl nitrate. Both central and systemic administration of MK-801 significantly increased intake of 15% sucrose. Intraperitoneal injection of atropine abolished MK-801-induced increase in sucrose intake, whereas administration into the fourth ventricle had no effect. To determine whether augmentation of cholinergic tone produces an enhancement of food intake in the absence of MK-801, we tested the ability of cisapride, a gastric prokinetic agent that promotes acetylcholine release through an action on presynaptic serotonin (5-HT4) receptors, to increase sucrose consumption. Cisapride (500 microg/kg ip) induced a small but significant increase in 15% sucrose intake (15.5 +/- 0.5 ml) compared with NaCl (13.0 +/- 0.6 ml). Furthermore, when MK-801 (100 microg/kg ip) was given in combination with cisapride, intake was significantly higher (19.8 +/- 0.9 ml) than following either agent given alone. Pretreatment with atropine abolished the cisapride-induced increase in intake (12.1 +/- 0.9 ml) as well as the increased intake induced by combining MK-801 and cisapride. These results suggest that blockade of NMDA-gated ion channels in the hindbrain increases food intake, in part, via a peripheral muscarinic cholinergic mechanism.     

Cardiovascular responses to substance P in the nucleus tractus solitarii: microinjection study in conscious rats

The cardiovascular effects of substance P (SP) microinjections in the nucleus tractus solitarii (NTS) were evaluated in conscious rats. We chose this model because it is an effective way to access some of the cardiovascular effects of neurotransmitters in the NTS without the inconvenience of blunting pathways with anesthetic agents or removing forebrain projections by decerebration. The cardiovascular responses to SP injections were also evaluated after chronic nodose ganglionectomy. We found that, in conscious rats, SP microinjections into the NTS induced hypertension and tachycardia. Unilateral and bilateral SP injections into the NTS caused a slow increase in blood pressure and heart rate that peaked 1.5-5 min after injection and lasted for 20-30 min. Nodose ganglionectomy increased the duration of the pressor and tachycardic effects of SP and enhanced the pressor response. These data show that SP in the NTS is involved in pressor pathways. The supersensitivity to SP seen after nodose ganglionectomy suggests that vagal afferent projections are involved in those pressor pathways activated by SP in the NTS.     

Secretin activates vagal primary afferent neurons in the rat: evidence from electrophysiological and immunohistochemical studies

In this study, we evaluated the vagal afferent response to secretin at physiological concentrations and localized the site of secretin's action on vagal afferent pathways in the rat. The discharge of sensory neurons supplying the gastrointestinal tract was recorded from nodose ganglia. Of 91 neurons activated by electrical vagal stimulation, 19 neurons showed an increase in firing rate in response to intestinal perfusion of 5-HT (from 1.5 +/- 0.2 to 25 +/- 4 impulses/20 s) but no response to intestinal distension. A close intra-arterial injection of secretin (2.5 and 5.0 pmol) elicited responses in 15 of these 19 neurons (from 1.5 +/- 0.2 impulses/20 s at basal to 21 +/- 4 and 43 +/- 5 impulses/20 s, respectively). Subdiaphragmatic vagotomy and perivagal application of capsaicin, but not supranodose vagotomy, completely abolished the secretin-elicited vagal nodose neuronal response. In a separate study, 9 tension receptor afferents among 91 neurons responded positively to intestinal distension but failed to respond to luminal 5-HT. These nine neurons also showed no response to administration of secretin. As expected, immunohistochemical studies showed that secretin administration significantly increased the number of Fos-positive neurons in vagal nodose ganglia. In conclusion, we demonstrated for the first time that vagal sensory neurons are activated by secretin at physiological concentrations. A subpopulation of secretin-sensitive vagal afferent fibers is located in the intestinal mucosa, many of which are responsive to luminal 5-HT.     

Intracerebroventricular administration of MK-801 increases food intake through mechanisms independent of gastric emptying

Systemic or hindbrain administration of MK-801, a noncompetitive N-methyl-D-aspartate receptor antagonist, increases meal size. To examine whether MK-801 enhances intake by increasing gastric emptying, we administered MK-801 (2.0 microg/3.0 microl) into the fourth ventricle [intracerebroventricular (ICV)] and measured feeding and gastric emptying of 5-ml NaCl or 15% sucrose loads. In a parallel experiment, we examined food intake and gastric emptying following intraperitoneal (IP) injection of MK-801 (100 microg/kg). MK-801, either IP or ICV, increased 30-min sucrose intake compared with control (12.3 +/- 0.7 vs. 9.8 +/- 0.5 and 16.6 +/- 2.0 vs. 10.7 +/- 0.7 ml, for IP and ICV administration, respectively). Also, IP MK-801 increased 5-min gastric emptying of NaCl (4.13 +/- 0.1 ml emptied) and sucrose (3.11 +/- 0.1 ml emptied) compared with control (3.75 +/- 0.2 and 2.28 +/- 0.1 ml emptied for NaCl and sucrose loads, respectively). In contrast, ICV MK-801 did not alter NaCl emptying (3.82 +/- 0.1 ml emptied) compared with control (3.82 +/- 0.3 ml emptied) and actually reduced gastric emptying of sucrose (2.1 +/- 0.2 and 2.94 +/- 0.1 ml emptied, for MK and vehicle, respectively). These data confirm previous results that systemic as well as hindbrain injection of MK-801 increases food intake. However, because ICV MK-801 failed to increase gastric emptying, these results indicate that MK-801 increases food intake through mechanisms independent of altered gastric emptying.     

Vanilloid receptor (VR1) expression in vagal afferent neurons innervating the gastrointestinal tract

The vanilloid receptor VR1 is a nonselective cation channel activated by capsaicin as well as increases in temperature and acidity, and can be viewed as molecular integrator of chemical and physical stimuli that elicit pain. The distribution of VR1 receptors in peripheral and central processes of rat primary vagal afferent neurons innervating the gastrointestinal tract was investigated by immunohistochemistry. Forty-two percent of neurons in the nodose ganglia retrogradely labeled from the stomach wall expressed low to moderate VR1 immunoreactivity (VR1-IR). VR1-IR was considerably lower in the nodose ganglia as compared to the jugular and dorsal root ganglia. In the vagus nerve, strongly VR1-IR fibers ran in separate fascicles that supplied mainly cervical and thoracic targets, leaving only weakly VR1-IR fibers in the subdiaphragmatic portion. Vagal afferent intraganglionic laminar endings (IGLEs) in the gastric and duodenal myenteric plexus did not express VR1-IR. Similarly, VR1-IR was contained in fibers running in perfect register with vagal afferents, but was not colocalized with horseradish peroxidase in the same varicosities of intramuscular arrays (IMAs) and vagal afferent fibers in the duodenal submucosa anterogradely labeled from the nodose ganglia. Only in the gastric mucosa did we find evidence for colocalization of VR1-IR in vagal afferent terminals. In contrast, many nerve fibers coursing through the myenteric and submucosal plexuses contained detectable VR1-IR, the majority of which colocalized calcitonin gene-related peptide immunoreactivity. In the dorsal medulla there was a dense plexus of VR1-IR varicose fibers in the commissural, dorsomedial and gelatinosus subnuclei of the medial NTS and the lateral aspects of the area postrema, which was substantially reduced, but not eliminated on the ipsilateral side after supranodose vagotomy. It is concluded that about half of the vagal afferents innervating the gastrointestinal tract express low levels of VR1-IR, but that presence in most of the peripheral terminal structures is below the immunohistochemical detection threshold.     

Vagal innervation of the rat pylorus: an anterograde tracing study using carbocyanine dyes and laser scanning confocal microscopy

In an attempt to identify the distribution and structure of vagal fibers and terminals in the gastroduodenal junction, vagal efferents were labeled in vivo by multiple injections of the fluorescent carbocyanine dye DiA into the dorsal motor nucleus (dmnX), and vagal afferents were anterogradely labeled by injections of DiI into the nodose ganglia of the same or separate rats. Thick frontal cryostat sections were analysed either with conventional or laser scanning confocal microscopy, using appropriate filter combinations and/or different wavelength laser excitation to distinguish the fluorescent tracers. Vagal efferent terminal-like structures were present in small ganglia within the circular sphincter muscle, which, in the absence of a well-developed, true myenteric plexus at this level, represent the myenteric ganglia. Furthermore, vagal efferent terminals were also present in submucosal ganglia, but were absent from mucosa, Brunner's glands and circular muscle fibers. Vagal afferent fibers and terminal-like structures were more abundant than efferents. The most prominent afferent terminals were profusely branching, large net-like aggregates of varicose fibers running within the connective tissue matrix predominantly parallel to the circular sphincter muscle bundles. Profusely arborizing, highly varicose endings were also present in large myenteric ganglia of the antrum and duodenum, in the modified intramuscular ganglia, and in submucosal ganglia. Additionally, afferent fibers and terminals were present throughout the mucosal lining of the gastroduodenal junction. The branching patterns of some vagal afferents suggested that individual axons produced multiple collaterals in different compartments. NADPH-diaphorase positive, possibly nitroxergic neurons were present in myenteric ganglia of the immediately adjacent antrum and duodenum, and fine varicose fibers entered the sphincter muscle from both sides, delineating the potential vagal inhibitory postganglionic innervation. These morphological results support the view of a rich and differentiated extrinsic neural control of this important gut region as suggested by functional studies.Abbreviations BSA Bovine serum albumin - CGRP calcitonin generelated peptide - DiA carbocyanine dye A - DiI carbocyanine dye I - dmnX dorsal motor nucleus of vagus - DMSO dimethylsulfoxide - ENK enkephalin - FITC fluorescin isothiocyanate - NADPH diaphorase nicotinamide adenine diphosphate - NPY neuropeptide Y - NTS nucleus tractus solitarii - PBS phosphate-buffered saline - VIP vasoactive intestinal peptide - WGA-HRP wheat-germ agglutinine-horseradish peroxidase     

Reduction of food intake by cholecystokinin requires activation of hindbrain NMDA-type glutamate receptors

Intraperitoneal injection of CCK reduces food intake and triggers a behavioral pattern similar to natural satiation. Reduction of food intake by CCK is mediated by vagal afferents that innervate the stomach and small intestine. These afferents synapse in the hindbrain nucleus of the solitary tract (NTS) where gastrointestinal satiation signals are processed. Previously, we demonstrated that intraperitoneal (IP) administration of either competitive or noncompetitive N-methyl-d-aspartate (NMDA) receptor antagonists attenuates reduction of food intake by CCK. However, because vagal afferents themselves express NMDA receptors at both central and peripheral endings, our results did not speak to the question of whether NMDA receptors in the brain play an essential role in reduction of feeding by CCK. We hypothesized that activation of NMDA receptors in the NTS is necessary for reduction of food intake by CCK. To test this hypothesis, we measured food intake following IP CCK, subsequent to NMDA receptor antagonist injections into the fourth ventricle, directly into the NTS or subcutaneously. We found that either fourth-ventricle or NTS injection of the noncompetitive NMDA receptor antagonist MK-801 was sufficient to inhibit CCK-induced reduction of feeding, while the same antagonist doses injected subcutaneously did not. Similarly fourth ventricle injection of d-3-(2-carboxypiperazin-4-yl)-1-propenyl-1-phosphoric acid (d-CPPene), a competitive NMDA receptor antagonist, also blocked reduction of food intake following IP CCK. Finally, d-CPPene injected into the fourth ventricle attenuated CCK-induced expression of nuclear c-Fos immunoreactivity in the dorsal vagal complex. We conclude that activation of NMDA receptors in the hindbrain is necessary for the reduction of food intake by CCK. Hindbrain NMDA receptors could comprise a critical avenue for control and modulation of satiation signals to influence food intake and energy balance.     

Effects of Excitatory Amino Acids on Cerebral Oxygen Consumption and Blood Flow in Rat

This investigation tested the importance of excitatory amino acids' effects on regional cerebral O₂ consumption and the concomitant changes in cerebral blood flow (rCBF) in isoflurane anesthetized rats. In the glutamate or N-methyl-D-aspartate (NMDA) groups, 10^–2 M glutamate or NMDA was topically applied to the right cortex and the left cortex was used as a control. One mg/kg dizocilpine maleate (MK-801), a non-competitive NMDA receptor antagonist, was administered (iv) to the MK-801 group and saline was given to the control group. Cortical rCBF was determined using ¹⁴C-iodoantipyrine and regional O₂ extraction was measured microspectrophotometrically. Cerebral O₂ consumption increased 77% after glutamate (contralateral cortex: 9.0 ± 1.1 ml O₂/min/100 g, glutamate treated cortex: 15.9 ± 3.9), while a 46% increase was observed with the same concentration of NMDA (contralateral cortex: 9.8 ± 2.0, NMDA treated cortex: 14.3 ± 5.5). After MK-801, the O₂ consumption decreased to 37% of the control value (control cortex: 7.0 ± 1.3, MK-801 treated cortex: 2.6 ± 3.9). MK-801 significantly decreased cerebral O₂ extraction from 7.1 ± 1.3 ml O₂/100 ml (control cortex) to 5.3 ± 0.6 (MK-801 treated cortex). However, there was no significant difference in cerebral O₂ extraction between treated and contralateral cortex in either the glutamate or NMDA groups. The increase in O₂ consumption caused by glutamate or NMDA was coupled with increased rCBF. Glutamate increased rCBF from 95 ± 5 ml/min/100 g (contralateral cortex) to 165 ± 31 (treated cortex), while NMDA increased rCBF from 114 ± 12 (contralateral cortex) to 178 ± 60 (treated cortex). MK-801 decreased O₂ consumption with a lesser decrease of rCBF. The rCBF was 48 ± 9 in the MK-801 treated cortex and 99 ± 22 in the control cortex. Some substances produced by the activation of NMDA receptors may be related to the coupling of cerebral metabolism and blood flow, since after blockade of NMDA receptors with MK-801, this relationship is uncoupled. These findings suggest that glutamatergic processes have a major effect on cerebral O₂ consumption and that this is at least partly due to NMDA receptors.     

NMDA receptor participation in control of food intake by the stomach

We previously reported that MK-801 (dizocilpine), an antagonist of N-methyl-D-aspartate (NMDA)-type glutamate receptors, increased meal size and duration in rats. MK-801 did not increase sham feeding or attenuate reduction of sham feeding by intraintestinal nutrient infusions. These results suggested that the MK-801-induced increase in meal size did not depend on antagonism of postgastric satiety signals. Consequently, we hypothesized that the NMDA antagonist might increase food intake by directly antagonizing gastric mechanosensory signals or by accelerating gastric emptying, thereby reducing gastric mechanoreceptive feedback. To test this hypothesis, we recorded intake of 15% sucrose in rats implanted with pyloric cuffs that could be closed to prevent gastric emptying. Sucrose intake was increased when the pyloric cuffs were open, allowing the stomach to empty. However, intake was not increased when the pyloric cuffs were inflated, causing gastric retention of all ingested sucrose. Direct measurements of gastric emptying revealed that MK-801 accelerated the emptying of 5-ml loads of 0.9% NaCl and 15% sucrose. Furthermore, MK-801 also accelerated the rate of emptying of freely ingested sucrose regardless of the volume ingested. Taken together with our previous findings, these results indicate that blockade of NMDA receptors with MK-801 does not increase food intake by antagonizing gastric mechanosensation. Rather, it accelerates gastric emptying, and thereby may indirectly reduce gastric mechanoreceptive cues, resulting in prolongation of eating. Modulation of gastric emptying rate by NMDA receptors could play an important role in the control of meal sizes.     

Targeted disruption of the murine CCK1 receptor gene reduces intestinal lipid-induced feedback inhibition of gastric function

Cholecystokinin (CCK), acting at CCK1 receptors (CCK1Rs) on intestinal vagal afferent terminals, has been implicated in the control of gastrointestinal function and food intake. Using CCK1R(-/-) mice, we tested the hypothesis that lipid-induced activation of the vagal afferent pathway and intestinal feedback of gastric function is CCK1R dependent. In anesthetized CCK1R(+/+) ("wild type") mice, meal-stimulated gastric acid secretion was inhibited by intestinal lipid infusion; this was abolished in CCK1R(-/-) mice. Gastric emptying of whole egg, measured by nuclear scintigraphy in awake mice, was significantly faster in CCK1R(-/-) than CCK1R(+/+) mice. Gastric emptying of chow was significantly slowed in response to administration of CCK-8 (22 pmol) in CCK1R(+/+) but not CCK1R(-/-) mice. Activation of the vagal afferent pathway was measured by immunohistochemical localization of Fos protein in the nucleus of the solitary tract (NTS; a region where vagal afferents terminate). CCK-8 (22 pmol ip) increased neuronal Fos expression in the NTS of fasted CCK1R(+/+) mice; CCK-induced Fos expression was reduced by 97% in CCK1R(-/-) compared with CCK1R(+/+) mice. Intralipid (0.2 ml of 20% Intralipid and 0.04 g lipid), but not saline, gavage increased Fos expression in the NTS of fasted CCK1R(+/+) mice; lipid-induced Fos expression was decreased by 47% in CCK1R(-/-) compared with CCK1R(+/+)mice. We conclude that intestinal lipid activates the vagal afferent pathway, decreases gastric acid secretion, and delays gastric emptying via a CCK1R-dependent mechanism. Thus, despite a relatively normal phenotype, intestinal feedback in response to lipid is severely impaired in these mice.     

Peripheral versus central modulation of gastric vagal pathways by metabotropic glutamate receptor 5

Metabotropic glutamate receptors (mGluR) are classified into group I, II, and III mGluR. Group I (mGluR1, mGluR5) are excitatory, whereas group II and III are inhibitory. mGluR5 antagonism potently reduces triggering of transient lower esophageal sphincter relaxations and gastroesophageal reflux. Transient lower esophageal sphincter relaxations are mediated via a vagal pathway and initiated by distension of the proximal stomach. Here, we determined the site of action of mGluR5 in gastric vagal pathways by investigating peripheral responses of ferret gastroesophageal vagal afferents to graded mechanical stimuli in vitro and central responses of nucleus tractus solitarius (NTS) neurons with gastric input in vivo in the presence or absence of the mGluR5 antagonist 2-methyl-6-(phenylethynyl)pyridine (MPEP). mGluR5 were also identified immunohistochemically in the nodose ganglia and NTS after extrinsic vagal inputs had been traced from the proximal stomach. Gastroesophageal vagal afferents were classified as mucosal, tension, or tension-mucosal (TM) receptors. MPEP (1-10 microM) inhibited responses to circumferential tension of tension and TM receptors. Responses to mucosal stroking of mucosal and TM receptors were unaffected. MPEP (0.001-10 nmol icv) had no major effect on the majority of NTS neurons excited by gastric distension or on NTS neurons inhibited by distension. mGluR5 labeling was abundant in gastric vagal afferent neurons and sparse in fibers within NTS vagal subnuclei. We conclude that mGluR5 play a prominent role at gastroesophageal vagal afferent endings but a minor role in central gastric vagal pathways. Peripheral mGluR5 may prove a suitable target for reducing mechanosensory input from the periphery, for therapeutic benefit.     

Gastric distension-induced release of 5-HT stimulates c-fos expression in specific brain nuclei via 5-HT3 receptors in conscious rats

We examined c-fos expression in specific brain nuclei in response to gastric distension and investigated whether 5-HT released from enterochromaffin (EC) cells was involved in this response. The role of 5-HT3 receptors in this mechanism was also addressed. Release of 5-HT was examined in an ex vivo-perfused stomach model, whereas c-fos expression in brain nuclei induced by gastric distension was examined in a freely moving conscious rat model. Physiological levels of gastric distension stimulated the vascular release of 5-HT more than luminal release of 5-HT, and induced c-fos expression in the nucleus of the solitary tract (NTS), area postrema (AP), paraventricular nucleus (PVN), and supraoptic nucleus (SON). The c-fos expression in all these brain nuclei was blocked by truncal vagotomy as well as by perivagal capsaicin treatment, suggesting that vagal afferent pathways may mediate this response. Intravenous injection of 5-HT3 receptor antagonist granisetron blocked c-fos expression in all brain nuclei examined, although intracerebroventricular injection of granisetron had no effect, suggesting that 5-HT released from the stomach may activate 5-HT3 receptors located in the peripheral vagal afferent nerve terminals and then induce brain c-fos expression. c-fos Positive cells in the NTS were labeled with retrograde tracer fluorogold injected in the PVN, suggesting that neurons in the NTS activated by gastric distension project axons to the PVN. The present results suggest that gastric distension stimulates 5-HT release from the EC cells and the released 5-HT may activate 5-HT3 receptors located on the vagal afferent nerve terminals in the gastric wall leading to neuron activation in the NTS and AP and subsequent activation of neurons in the PVN and SON.     

D-glucose modulates synaptic transmission from the central terminals of vagal afferent fibers

Experimental evidence suggests that glucose modulates gastric functions via vagally mediated effects. It is unclear whether glucose affects only peripheral vagal nerve activity or whether glucose also modulates vagal circuitry at the level of the brain stem. This study used whole cell patch-clamp recordings from neurons of the nucleus of the tractus solitarius (NTS) to assess whether acute variations in glucose modulates vagal brain stem neurocircuitry. Increasing D-glucose concentration induced a postsynaptic response in 40% of neurons; neither the response type (inward vs. outward current) nor response magnitude was altered in the presence of tetrodotoxin suggesting direct effects on the NTS neuronal membrane. In contrast, reducing d-glucose concentration induced a postsynaptic response (inward or outward current) in 54% of NTS neurons; tetrodotoxin abolished these responses, suggesting indirect sites of action. The frequency, but not amplitude, of spontaneous and miniature excitatory postsynaptic currents (EPSCs) was correlated with d-glucose concentration in 79% of neurons tested (n = 48). Prior surgical afferent rhizotomy abolished the ability of D-glucose to modulate spontaneous EPSC frequency, suggesting presynaptic actions at vagal afferent nerve terminals to modulate glutamatergic synaptic transmission. In experiments in which EPSCs were evoked via electrical stimulation of the tractus solitarius, EPSC amplitude correlated with D-glucose concentration. These effects were not mimicked by L-glucose, suggesting the involvement of glucose metabolism, not uptake, in the nerve terminal. These data suggest that the synaptic connections between vagal afferent nerve terminals and NTS neurons are a strong candidate for consideration as one of the sites where glucose-evoked changes in vagovagal reflexes occurs.     

Enhanced NMDA Receptor-Mediated Modulation of Excitatory Neurotransmission in the Dorsal Vagal Complex of Streptozotocin-Treated,Chronically Hyperglycemic Mice

A variety of metabolic disorders, including complications experienced by diabetic patients, have been linked to altered neural activity in the dorsal vagal complex. This study tested the hypothesis that augmentation of N-Methyl-D-Aspartate (NMDA) receptor-mediated responses in the vagal complex contributes to increased glutamate release in the dorsal motor nucleus of the vagus nerve (DMV) in mice with streptozotocin-induced chronic hyperglycemia (i.e., hyperglycemic mice), a model of type 1 diabetes. Antagonism of NMDA receptors with AP-5 (100 μM) suppressed sEPSC frequency in vagal motor neurons recorded in vitro, confirming that constitutively active NMDA receptors regulate glutamate release in the DMV. There was a greater relative effect of NMDA receptor antagonism in hyperglycemic mice, suggesting that augmented NMDA effects occur in neurons presynaptic to the DMV. Effects of NMDA receptor blockade on mEPSC frequency were equivalent in control and diabetic mice, suggesting that differential effects on glutamate release were due to altered NMDA function in the soma-dendritic membrane of intact afferent neurons. Application of NMDA (300 μM) resulted in greater inward current and current density in NTS neurons recorded from hyperglycemic than control mice, particularly in glutamatergic NTS neurons identified by single-cell RT-PCR for VGLUT2. Overall expression of NR1 protein and message in the dorsal vagal complex were not different between the two groups. Enhanced postsynaptic NMDA responsiveness of glutamatergic NTS neurons is consistent with tonically-increased glutamate release in the DMV in mice with chronic hyperglycemia. Functional augmentation of NMDA-mediated responses may serve as a physiological counter-regulatory mechanism to control pathological disturbances of homeostatic autonomic function in type 1 diabetes.     

Acetylcholine content in superior cervical ganglion following reinnervation of vagal afferent fibers in cat

The superior cervical ganglion (SCG) was reinnervated by vagal afferent fibers by cross anastomosis between the cranial end of nodose ganglion and the caudal end of SCG in cats. Formation of functional synapses was evidenced by unilateral mydriasis and contraction of the nictitating membrane in response to inflation of the stomach with a balloon or to electrical stimulation of the afferent vagus. The acetylcholine (ACh) content in the cross-anastomosed SCG (reinnervated by vagal afferent fibers) was measured. In anastomosed SCG, the ACh content was about half of normal SCG, but significantly higher than chronically decentralized SCG. Also the ACh content in nodose ganglion (NDG) was investigated in situations in which there was anastomosis, chronic supra, infra, or supra-/infranodose vagotomy. The ACh content of anastomosed NDG was near that of supranosdose vagotomized ganglion. The ACh content of supra-/infranodose vagotomized NDG, which can be considered the NDG itself, was as much as that of normal intact NDG. It was found that the ACh content of infranodose vagotomized NDG was increased, possibly the result of vagal efferent axonal flow or transport. The ACh content of vagal trunk with or without infranodose vagotomy was also measured. The ACh content of vagal trunk with infranodose vagotomy was smaller than that of the normal trunk, but there was still a considerable quantity of ACh. There was no significant change in wet weight of the SCG and NDG before or after the operations. From these results we have concluded that the transmission of the cross-anastomosed SCG (reinnervated with vagal afferent nerve) was cholinergic; and that the vagal afferent nerve have afferent cell bodies not only in NDG but also in peripheral vagal trunks (infranodose portion). These results strongly suggest that vagal afferent fibers are in part cholinergic.     

Role of CCK1 and Y2 receptors in activation of hindbrain neurons induced by intragastric administration of bitter taste receptor ligands

G-protein-coupled receptors signaling bitter taste (T2Rs) in the oral gustatory system and the alpha-subunit of the taste-specific G-protein gustducin are expressed in the gastrointestinal (GI) tract. alpha-Subunit of the taste-specific G-protein gustducin colocalizes with markers of enteroendocrine cells in human and mouse GI mucosa, including peptide YY. Activation of T2Rs increases cholecystokinin (CCK) release from the enteroendocrine cell line, STC-1. The aim of this study was to determine whether T2R agonists in the GI tract activate neurons in the nucleus of the solitary tract (NTS) and whether this activation is mediated by CCK and peptide YY acting at CCK(1) and Y(2) receptors. Immunocytochemistry for the protooncogene c-Fos protein, a marker for neuronal activation, was used to determine activation of neurons in the midregion of the NTS, the region where vagal afferents from the GI tract terminate. Intragastric administration of the T2R agonist denatonium benzoate (DB), or phenylthiocarbamide (PTC), or a combination of T2R agonists significantly increased the number of Fos-positive neurons in the mid-NTS; subdiaphragmatic vagotomy abolished the NTS response to the mixture of T2R agonists. Deletion of CCK(1) receptor gene or blockade of CCK(1) receptors with devazepide abolishes the activation of NTS neurons in response to DB, but had no effect on the response to PTC. Administration of the Y(2) receptor antagonist BIIE0246 blocks the activation of NTS neurons to DB, but not PTC. These findings suggest that activation of neurons in the NTS following administration of T2R agonists to the GI tract involves CCK(1) and Y(2) receptors located on vagal afferent terminals in the gut wall. T2Rs may regulate GI function via release of regulatory peptides and activation of the vagal reflex pathway.     

Leptin and CCK modulate complementary background conductances to depolarize cultured nodose neurons

We have previously reported that intraceliac infusion of leptin induces a reduction of meal size that depends on intact vagal afferents. This effect of leptin is enhanced in the presence of cholecystokinin (CCK). The mechanisms by which leptin and CCK activate vagal afferent neurons are not known. In the present study, we have begun to address this question by using patch-clamp electrophysiological techniques to examine the mechanisms by which leptin and CCK activate cultured vagal afferents from adult rat nodose ganglia. We found that leptin depolarized 41 (60%) of 68 neurons. The magnitude of membrane depolarization was dependent on leptin concentration and occurred in both capsaicin-sensitive and capsaicin-insensitive neurons. We also found that a majority (16 of 22; 73%) of nodose neurons activated by leptin were also sensitive to CCK. CCK-induced depolarization was primarily associated with the increase of an inward current (11 of 12), whereas leptin induced multiple changes in background conductances through a decrease in an outward current (7 of 13), an increase in an inward current (3 of 13), or both (3 of 13). However, further isolation of background currents by recording in solutions that contained only sodium or only potassium revealed that both leptin and CCK were capable of increasing a sodium-dependent conductance or inhibiting a potassium-dependent conductance. Our results support the hypothesis that vagal afferents are a point of convergence and integration of leptin and CCK signaling for control of food intake and suggest multiple ionic mechanisms by which leptin and CCK activate vagal afferent neurons. cholecystokinin; vagal afferents; capsaicin; satiation     

Glucose increases synaptic transmission from vagal afferent central nerve terminals via modulation of 5-HT3 receptors

Acute hyperglycemia has profound effects on vagally mediated gastrointestinal functions. We have reported recently that the release of glutamate from the central terminals of vagal afferent neurons is correlated directly with the extracellular glucose concentration. The present study was designed to test the hypothesis that 5-HT(3) receptors present on vagal afferent nerve terminals are involved in this glucose-dependent modulation of glutamatergic synaptic transmission. Whole-cell patch-clamp recordings were made from neurons of the nucleus tractus solitarius (NTS) in thin rat brainstem slices. Spontaneous and evoked glutamate release was decreased in a concentration-dependent manner by the 5-HT(3) receptor selective antagonist, ondansetron. Alterations in the extracellular glucose concentration induced parallel shifts in the ondansetron-mediated inhibition of glutamate release. The changes in excitatory synaptic transmission induced by extracellular glucose concentration were mimicked by the serotonin uptake inhibitor, fenfluramine. These data suggest that glucose alters excitatory synaptic transmission within the rat brainstem via actions on tonically active 5-HT(3) receptors, and the number of 5-HT(3) receptors on vagal afferent nerve terminals is positively correlated with the extracellular glucose concentration. These data indicate that the 5-HT(3) receptors present on synaptic connections between vagal afferent nerve terminals and NTS neurons are a strong candidate for consideration as one of the sites where glucose acts to modulate vagovagal reflexes.