Acute high-fat diet upregulates glutamatergic signaling in the dorsal motor nucleus of the vagus

Abstract

Obesity is associated with dysregulation of vagal neurocircuits controlling gastric functions, including food intake and energy balance. In the short term, however, caloric intake is regulated homeostatically although the precise mechanisms responsible are unknown. The present study examined the effects of acute high-fat diet (HFD) on glutamatergic neurotransmission within central vagal neurocircuits and its effects on gastric motility. Sprague-Dawley rats were fed a control or HFD diet (14% or 60% kcal from fat, respectively) for 3-5 days. Whole cell patch-clamp recordings and brainstem application of antagonists were used to assess the effects of acute HFD on glutamatergic transmission to dorsal motor nucleus of the vagus (DMV) neurons and subsequent alterations in gastric tone and motility. After becoming hyperphagic initially, caloric balance was restored after 3 days following HFD exposure. In control rats, the non- N-methyl-d-aspartate (NMDA) receptor antagonist, 6,7-dinitroquinoxaline-2,3-dione (DNQX), but not the NMDA receptor antagonist, amino-5-phosphonopentanoate (AP5), significantly decreased excitatory synaptic currents and action potential firing rate in gastric-projecting DMV neurons. In contrast, both AP5 and DNQX decreased excitatory synaptic transmission and action potential firing in acute HFD neurons. When microinjected into the brainstem, AP5, but not DNQX, decreased gastric motility and tone in acute HFD rats only. These results suggest that acute HFD upregulates NMDA receptor-mediated currents, increasing DMV neuronal excitability and activating the vagal efferent cholinergic pathway, thus increasing gastric tone and motility. Although such neuroplasticity may be a persistent adaptation to the initial exposure to HFD, it may also be an important mechanism in homeostatic regulation of energy balance. NEW & NOTEWORTHY Vagal neurocircuits are critical to the regulation of gastric functions, including satiation and food intake. Acute high-fat diet upregulates glutamatergic signaling within central vagal neurocircuits via activation of N-methyl-d-aspartate receptors, increasing vagal efferent drive to the stomach. Although it is possible that such neuroplasticity is a persistent adaptation to initial exposure to the high-fat diet, it may also play a role in the homeostatic control of feeding.

Keywords: N-methyl-d-aspartate; brainstem; glutamate; high-fat diet; vagus.

Figures

Fig. 1.

Restoration of caloric balance occurs within 3 days of exposure to a high-fat diet (HFD). A: graphical illustration of the experimental timeline. B: graphical summary of caloric intake following exposure to an HFD. Single-housed rats were fed a control diet and, after acclimation, continued on a control diet (n = 4) or fed an HFD (n = 17). Rats were weighed daily, and their total food intake was calculated. Note that rats became hyperphagic and consumed a larger number of calories immediately upon exposure to the HFD (day 1). By day 3 onward, however, energy balance had been restored, and there were no differences in caloric intake between control and HFD-fed rats. *P < 0.05 (Student’s paired t-test). C: graphical summary of caloric intake following exposure to an HFD, expressed as a percentage of averaged baseline control intake. Note that caloric intake increased in 16/17 rats after 1 day of food intake but was restored to within 25% of baseline levels by 2 days in 7/16 rats, and by 3–5 days in the remaining 9/16 rats. Mean values for each day are marked by gray horizontal lines; the black dashed line indicates 125% of baseline caloric intake.

Fig. 2.

The N-methyl-d-aspartate (NMDA) receptor-selective antagonist, amino-5-phosphonopentanoate (AP5), decreases action potential (AP) firing rate in acute high-fat diet (HFD), but not control, dorsal motor nucleus of the vagus (DMV) neurons. A: representative traces from a gastric-projecting DMV neuron from a control diet rat, current clamped at a potential that allowed AP firing of ~1 Hz. Perfusion with AP5 (25 μM) had no effect on AP firing rate in 7/10 neurons tested (3 rats). Subsequent application of 6,7-dinitroquinoxaline-2,3-dione (DNQX) (30 μM) decreased AP firing rate in 8/10 neurons tested (3 rats). B: representative traces from a gastric-projecting DMV neuron from an acute HFD rat, current clamped at a potential to allow AP firing of ~1 Hz. Perfusion with AP5 (25 μM) decreased AP firing in all 5 neurons (from 3 rats) tested. Subsequent application of DNQX (30 μM) did not affect AP firing rate further. C: graphical summary of the responses of control (left; n = 10 neurons from 3 rats) and acute HFD (right; n = 5 neurons from 3 rats) gastric-projecting DMV neurons to perfusion with AP5 and DNQX. Note that AP5 decreased the firing rate in all acute HFD neurons but in only a minority of control neurons. *P < 0.05 vs. baseline (Student’s paired t-test), #P < 0.05 vs. control (Student’s paired t-test). D: graphical summary of the proportion of gastric-projecting DMV neurons responding to AP5 with a decrease in AP firing rate.*P < 0.05 vs. control (χ2-test).

Fig. 3.

The N-methyl-d-aspartate receptor (NMDA-R)-selective antagonist, amino-5-phosphonopentanoate (AP5), decreases excitatory synaptic transmission to acute high-fat diet (HFD), but not control, dorsal motor nucleus of the vagus (DMV) neurons. A: 6 overlapping consecutive traces from a control gastric-projecting DMV neuron voltage clamped at −50 mV illustrating miniature excitatory postsynaptic currents (mEPSCs). Perfusion with AP5 (25 μM), had no effect on mEPSC frequency or amplitude in 17/18 neurons. Subsequent application of 6,7-dinitroquinoxaline-2,3-dione (DNQX) (30 μM) abolished all mEPSCs, confirming their glutamatergic nature. B: sample trace of mEPSCs in an acute HFD gastric-projecting DMV neuron, voltage clamped at −50 mV (6 overlapping consecutive traces). Perfusion with AP5 decreased mEPSC amplitude and frequency in 6/15 neurons; subsequent application of DNQX abolished all mEPSCs, confirming the role of both NMDA-R and non-NMDA-R (presumably α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor, AMPA-R) in synaptic glutamatergic currents. C: graphical summary of the effects of AP5 on mEPSC amplitude in control (left; n = 17, 8 rats) and acute HFD (right; n = 6 responding neurons and 9 nonresponding neurons, 8 rats) gastric-projecting DMV neurons. Neurons were classified as responsive based on whether there was a significant change in amplitude >25% from baseline. D: graphical summary of the effects of AP5 and DNQX on mEPSC frequency in control (left; n = 17 neurons, 8 rats) and acute HFD (right; n = 6 responding neurons and 9 nonresponding neurons, 8 rats) gastric-projecting DMV neurons. Neurons were classified as responsive based upon the ability of AP5 to decrease mEPSC amplitude. ● and ■ indicated response to AP5; ○ and □ indicate response to AP5 and DNQX. All mEPSCs were abolished by DNQX (control) or a combination of AP5 and DNQX (acute HFD). E: graphical summary of the effects of AP5 and DNQX on mEPSC charge transfer in control (left; n = 17 neurons, 8 rats) and acute HFD (right; n = 6 responding neurons and 9 nonresponding neurons, 8 rats) gastric-projecting DMV neurons. Neurons were classified as responsive based on the ability of AP5 to decrease mEPSC amplitude. ● and ■ indicate response to AP5; ○ and □ indicate response to AP5 and DNQX. *P < 0.05 vs. baseline (Student's paired t-test). #P < 0.05 vs. control (Student's paired t-test).

Fig. 4.

Acute high-fat diet (HFD) exposure alters the N-methyl-d-aspartate (NMDA): α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) ratio in dorsal motor nucleus of the vagus (DMV) neurons. Glutamate currents were evoked electrically via stimulation of the adjacent nucleus tractus solitarius (NTS) with a bipolar electrode. Gastric-projecting DMV neurons were voltage clamped at potentials between −80 mV and +40 mV in the absence and presence of the non-NMDA receptor (R)-selective antagonist, 6,7-dinitroquinoxaline-2,3-dione (DNQX) (30 μM) to reveal the proportion of NMDA-R-mediated currents at each potential. A: representative evoked excitatory postsynaptic currents (eEPSCs) in control (top, black) and acute HFD (bottom, gray) gastric-projecting DMV neurons at −80 mV and +40 mV, in the absence (left) and presence (right) of DNQX. Note that, in control DMV neurons, DNQX almost abolished the eEPSC at −80 mV, suggesting that NMDA-R activation contributed little, if at all, to the glutamatergic eEPSC at this potential, whereas AMPA-R activation contributed little to the eEPSC at +40 mV. In contrast, the effects of DNQX to inhibit the eEPSC at −80 mV were attenuated significantly in acute HFD DMV neurons, suggesting that NMDA-R activation contributed significantly to the glutamatergic current at this potential. B: magnitude of the NMDA-R-mediated current in control and acute HFD DMV neurons was calculated at holding potentials between −80 mV and +40 mV to create a current-voltage relationship. Note that, in acute HFD DMV neurons, the NMDA-R-mediated current is increased significantly at potentials negative to −40 mV, suggesting a significant contribution to glutamatergic-mediated synaptic currents at rest (n = 3–8 neurons from 3 to 4 rats, respectively), *P < 0.05 vs. control (Student’s t-test). C: graphical representation of NMDA-R-mediated (○) and AMPA-R-mediated (●) currents as a percentage of total eEPSC at potentials between −80 mV and +40 mV in control gastric-projecting DMV neurons (n = 3–8 neurons, 3 rats). D: graphical representation of NMDA-R-mediated (○) and AMPA-R-mediated (●) currents as a percentage of total eEPSC at potentials between −80 mV and +40 mV in acute HFD gastric-projecting DMV neurons (n = 3–8 neurons, 4 rats). E: graphical representation of AMPA-R-mediated currents as a percentage of total eEPSC at potentials between −80 mV and +40 mV in control (black) and acute HFD (gray) gastric-projecting DMV neurons (n = 3–8 neurons for each, 3–4 rats). *P < 0.05 vs. control (Student’s t-test). F: graphical representation of NMDA-R-mediated currents as a percentage of total eEPSC at potentials between −80 mV and +40 mV in control (black) and acute HFD (gray) gastric-projecting DMV neurons (n = 3–8 neurons for each, 3–4 rats; *P < 0.05 vs. control (Student’s t-test).

Fig. 5.

Brainstem microinjection of ionotropic glutamate receptor antagonists inhibits gastric tone and motility in acute high-fat diet (HFD) rats. A: representative recording demonstrating that dorsal vagal complex (DVC) microinjection of the nonselective glutamate receptor antagonist, kynurenic acid (100 pmol/60 nl) had no effect on antrum tone or motility in control diet rats (top). In contrast, DVC microinjection of kynurenic acid (100 pmol/60 nl; black trace), amino-5-phosphonopentanoate (AP5) (500 pmol/60 nl; red), but not 6,7-dinitroquinoxaline-2,3-dione (DNQX) (10 pmol/60 nl; blue), significantly inhibits antrum tone and motility in acute HFD rats. B: graphical representation of effects of brainstem microinjection of glutamate receptor antagonists on antrum motility (left) and tone (right) (n = 3–8 per data point; *P < 0.05 vs. baseline; paired Student’s t-test). C: map illustrating all brainstem microinjection sites, divided into rostral (top), intermediate (middle), and caudal (bottom) areas. Note that, for the sake of clarity, injection sites are marked bilaterally although all experiments were conducted using microinjections into the left DVC because recordings of motility and tone were made from the ventral stomach. D: photomicrograph illustrating a brainstem microinjection (arrow) at the level of the intermediate DVC. AP, area postrema; NTS, nucleus tractus solitarius; DMV, dorsal motor nucleus of the vagus; CC, central canal.

Fig. 6.

Brainstem microinjection of N-methyl-d-aspartate (NMDA) receptor antagonists inhibits gastric motility and tone in a vagally dependent manner involving the efferent cholinergic pathway. A: representative recordings demonstrating that the effects of dorsal vagal complex (DVC) microinjection of kynurenic acid (100 pmol/60 nl; top) are blocked by complete subdiaphragmatic vagotomy (Vgtx; right) and infusion of bethanechol (30 μg·kg−1·0.5 ml−1; middle) but not by infusion of nitro-l-arginine methyl ester (l-NAME) (10 mg·kg−1·0.5 ml−1; left). B: graphical summary of the effects of DVC microinjection of kynurenic acid on antrum motility before and after perfusion of l-NAME, perfusion of bethanecol, and complete subdiaphragmatic Vgtx, perfusion of l-NAME, and perfusion of bethanechol (n = 3–5 per data point; *P < 0.05 vs. baseline paired Student’s t-test). C: graphical summary of the effects of DVC microinjection of kynurenic acid on antrum tone, before and after complete subdiaphragmatic Vgtx, perfusion of l-NAME, and perfusion with bethanechol (n = 3–5 per data point; *P < 0.05 vs. baseline paired Student’s t-test).