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The hormone leptin reduces food intake through actions in the peripheral and central nervous systems, including in the hindbrain nucleus of the solitary tract (NTS). The NTS receives viscerosensory information via vagal afferents, including information from the gastrointestinal tract, which is then relayed to other central nervous system (CNS) sites critical for control of food intake. Leptin receptors (lepRs) are expressed by a subpopulation of NTS neurons, and knockdown of these receptors increases both food intake and body weight. Recently, we demonstrated that leptin increases vagal activation of lepR-expressing neurons via increased NMDA receptor (NMDAR) currents, thereby potentiating vagally evoked firing. Furthermore, chemogenetic activation of these neurons was recently shown to inhibit food intake. However, the vagal inputs these neurons receive had not been characterized. Here we performed whole cell recordings in brain slices taken from lepRCre × floxedTdTomato mice and found that lepR neurons of the NTS are directly activated by monosynaptic inputs from C-type afferents sensitive to the transient receptor potential vanilloid type 1 (TRPV1) agonist capsaicin. CCK administered onto NTS slices stimulated spontaneous glutamate release onto lepR neurons and induced action potential firing, an effect mediated by CCKR 1 . Interestingly, NMDAR activation contributed to the current carried by spontaneous excitatory postsynaptic currents (EPSCs) and enhanced CCK-induced firing. Peripheral CCK also increased c-fos expression in these neurons, suggesting they are activated by CCK-sensitive vagal afferents in vivo. Our results indicate that the majority of NTS lepR neurons receive direct inputs from CCK-sensitive C vagal-type afferents, with both peripheral and central CCK capable of activating these neurons and NMDARs able to potentiate these effects.

To investigate the role of glial cells in the regulation of glucoprivic responses in rats, a chemogenetic approach was used to activate astrocytes neighboring catecholamine (CA) neurons in the ventromedial medulla (VLM) where A1 and C1 CA cell groups overlap (A1/C1). Previous results indicate that activation of CA neurons in this region is necessary and sufficient for feeding and corticosterone release in response to glucoprivation. However, it is not known whether astrocyte neighbors of CA neurons contribute to glucoregulatory responses. Hence, we made nanoinjections of AAV5-GFAP-hM3D(Gq)-mCherry to selectively transfect astrocytes in the A1/C1 region with the excitatory designer receptor exclusively activated by designer drugs (DREADDs), hM3D(Gq). After allowing time for DREADD expression, we evaluated the rats for increased food intake and corticosterone release in response to low systemic doses of the antiglycolytic agent, 2-deoxy-d-glucose (2DG), alone and in combination with the hM3D(Gq) activator clozapine- n -oxide (CNO). We found that DREADD-transfected rats ate significantly more food when 2DG and CNO were coadministered than when either 2DG or CNO was injected alone. We also found that CNO significantly enhanced 2DG-induced FOS expression in the A1/C1 CA neurons, and that corticosterone release also was enhanced when CNO and 2DG were administered together. Importantly, CNO-induced activation of astrocytes in the absence of 2DG did not trigger food intake or corticosterone release. Our results indicate that during glucoprivation, activation of VLM astrocytes cells markedly increases the sensitivity or responsiveness of neighboring A1/C1 CA neurons to glucose deficit, suggesting a potentially important role for VLM astrocytes in glucoregulation.

Neuropeptide Y (NPY), peptide YY (PYY), and their cognate receptors (YR) are expressed by subpopulations of central and peripheral nervous system neurons. Intracerebroventricular injections of NPY or PYY increase food intake, and intrahypothalamic NPY1 or NPY5 receptor agonist injections also increase food intake. In contrast, injection of PYY in the periphery reduces food intake, apparently by activating peripheral Y2R. The dorsal vagal complex (DVC) of the hindbrain is the site where vagal afferents relay gut satiation signals to the brain. While contributions of the DVC are increasingly investigated, a role for DVC YR in control of food intake has not been examined systematically. We used in situ hybridization to confirm expression of Y1R and Y2R, but not Y5R, in the DVC and vagal afferent neurons. We found that nanoinjections of a Y2R agonist, PYY-(3-36), into the DVC significantly increased food intake over a 4-h period in satiated male rats. PYY-(3-36)-evoked food intake was prevented by injection of a selective Y2R antagonist. Injection of a Y1R/Y5R-preferring agonist into the DVC failed to increase food intake at doses reported to increase food intake following hypothalamic injection. Finally, injection of PYY-(3-36) into the DVC prevented reduction of 30-min food intake following intraperitoneal injection of cholecystokinin (CCK). Our results indicate that activation of DVC Y2R, unlike hypothalamic or peripheral Y2R, increases food intake. Furthermore, in the context of available electrophysiological observations, our results are consistent with the hypothesis that DVC Y2R control food intake by dampening vagally mediated satiation signals in the DVC.

Hindbrain injection of a melanocortin-3/4 receptor agonist, MTII, reduces food intake primarily by reducing meal size. Our previously reported results indicate that N-methyl-D-aspartate-type glutamate receptors (NMDAR) in the nucleus of the solitary tract (NTS) play an important role in the control of meal size and food intake. Therefore, we hypothesized that activation of NTS NMDARs contribute to reduction of food intake in response to fourth ventricle or NTS injection of MTII. We found that coinjection of a competitive NMDAR antagonist (d-CPP-ene) with MTII into the fourth ventricle or directly into the NTS of adult male rats attenuated MTII-induced reduction of food intake. Hindbrain NMDAR antagonism also attenuated MTII-induced ERK1/2 phosphorylation in NTS neurons and prevented synapsin I phosphorylation in central vagal afferent endings, both of which are cellular mechanisms previously shown to participate in hindbrain melanocortinergic reduction of food intake. Together, our results indicate that NMDAR activation significantly contributes to reduction of food intake following hindbrain melanocortin receptor activation, and it participates in melanocortinergic signaling in NTS neural circuits that mediate reduction of food intake., (Copyright © 2015 the American Physiological Society.)

Previous reports suggest that glucagon-like peptide (GLP-1), a peptide secreted from the distal small intestine, is an endocrine satiation signal. Nevertheless, there are conflicting reports regarding the site where circulating GLP-1 acts to reduce food intake. To test the hypothesis that vagal afferents are necessary for reduction of food intake by circulating GLP-1, we measured intake of 15% sucrose during intravenous GLP-1 infusion in intact, vagotomized, and capsaicin-treated rats. We also measured sucrose intake during intravenous infusion of cholecystokinin, a peptide known to reduce food intake via abdominal vagal afferents. We found that reduction of intake by GLP-1 was not diminished by capsaicin treatment or vagotomy. In fact, reduction of sucrose intake by our highest GLP-1 dose was enhanced in vagotomized and capsaicin-treated rats. Intravenous GLP-1 induced comparable increases of hindbrain c-Fos immunoreactivity in intact, capsaicin-treated, and vagotomized rats. Plasma concentrations of active GLP-1 in capsaicin-treated rats did not differ from those of controls during the intravenous infusions. Finally, capsaicin treatment was not associated with altered GLP-1R mRNA in the brain, but nodose ganglia GLP-1R mRNA was significantly reduced in capsaicin-treated rats. Although reduction of food intake by intraperitoneal cholecystokinin was abolished in vagotomized and capsaicin-treated rats, reduction of intake by intravenous cholecystokinin was only partially attenuated. These results indicate that vagal or capsaicin-sensitive neurons are not necessary for reduction of food intake by circulating (endocrine) GLP-1, or cholecystokinin. Vagal participation in satiation by these peptides may be limited to paracrine effects exerted near the sites of their secretion.

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.

We have previously shown that blockade of N-methyl-d-aspartate (NMDA) receptors in the caudal brain stem delays satiation and increases food intake. NMDA receptors are heterodimers made up of distinct, but different, ion channel subunits. The NR2 subunits of the NMDA receptor contain the binding site for glutamate. About half of vagal afferents express immunoreactivity for NMDA NR2B subunit and about half of the NR2B expressing afferents also express NMDA NR2C or NR2D subunits. This suggests that increased food intake may be evoked by interference with glutamate binding to NMDA channels containing the NR2B subunit. To test this, we measured deprivation-induced intake of 15% sucrose solution following fourth ventricle and intra-nucleus of the solitary tract (intra-NTS) injections of Conantokin G (Con G; NR2B blocker), d-3-(2-carboxypiperazin-4-yl)-1-propenyl-1-phosphoric acid (d-CPPene; NR2B/2A blocker), and (+/-)-cis-1-(phenanthren-2yl-carbonyl)piperazine-2,3-dicarboxylic acid (PPDA; NR2D/C blocker). Fourth ventricular administration of Con G (5, 20, 40, 80 ng), d-CPPene (3.0, 6.25, 12.5, 25, 50, 100 ng), and PPDA (300, 400 ng) increased sucrose intake significantly compared with control. Likewise, injections of Con G (10 ng), d-CPPene (5 ng, 10 ng), and PPDA (0.5, 1.0, 2.5, 5.0 ng) directly into the NTS significantly increased sucrose intake. These results show that hindbrain injection of competitive NMDA antagonists with selectivity or preference for the NMDA receptor NR2B or NR2C subunits increases food intake.

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.

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.

Imaging fluorescent measurements with fura 2 were used to examine cytosolic calcium signals induced by sulfated CCK octapeptide (CCK-8) in dissociated vagal afferent neurons from adult rat nodose ganglia. We found that 40% (184/465) of the neurons responded to CCK-8 with a transient increase in cytosolic calcium. The threshold concentration of CCK-8 for inducing the response varied from 0.01 to 100 nM. In most neurons (13/16) the response was eliminated by removing extracellular calcium. Depleting intracellular calcium stores with thapsigargin slightly augmented the response. Most neurons were unresponsive to nonsulfated CCK-8. The response was eliminated by the CCK-A receptor antagonist lorglumide. Low concentrations of JMV-180 had no effect; however, high concentrations of JMV-180 reduced responses to CCK-8. These results demonstrate that CCK acts at the low-affinity site of the CCK-A receptor to trigger the entry of extracellular calcium into vagal afferent neurons. Increased cytosolic calcium may participate in acute activation of vagal afferent neurons, or it may initiate long-term changes, which modulate future neuronal responses to sensory stimuli.

In the present study, we tested the hypothesis that a single daily injection of the gut peptide CCK, together with continuous leptin infusion, would produce significantly greater loss of body weight than leptin alone. We found that a single daily intraperitoneal injection of CCK-8 (0.5 microg/kg) significantly enhanced the weight-reducing effects of 0.5 microg/day leptin infused continuously into the lateral ventricle of male Sprague-Dawley rats by osmotic minipump. However, CCK and leptin together did not enhance reduction of daily chow intake. Furthermore, there was no synergistic reduction of 30-min sucrose intake, although a significant main effect of both leptin and CCK was observed on sucrose intake. These results 1) confirm our previous reports of synergy between leptin and CCK on body weight, 2) demonstrate that enhancement of leptin-induced weight loss does not require bolus administration of leptin, and 3) suggest that enhanced body weight loss following leptin and CCK does not require synergistic reduction of food intake by leptin and CCK.