Articles in PresS. Am J Physiol Regul Integr Comp Physiol (January 15, 2004). 10.1152/ajpregu.00570.2003
NMDA RECEPTOR BLOCKADE ATTENUATES CCK-INDUCED REDUCTION OF REAL FEEDING BUT NOT SHAM FEEDING
Mihai Covasa1, Robert C. Ritter2, and Gilbert A. Burns2 Department, of Nutritional Sciences, College of Health and Human Development, The Pennsylvania State University, University Park, PA, 16802-6504 and 2Department of Comparative Anatomy, Pharmacology and Physiology, Program in Neuroscience, Washington State University, Pullman, WA, 99163-6520 1
Abbreviated title: NMDA blockade and cholecystokinin
Address correspondence to: Mihai Covasa Department of Nutritional Sciences College of Health and Human Development The Pennsylvania State University 126 South Henderson University Park, PA, 16802 Email:
[email protected]
Copyright (c) 2004 by the American Physiological Society.
ABSTRACT Systemic injection of MK-801, a non-competitive 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 µg/kg, IP) reversed the inhibitory effects of CCK-8 (2 and 4 µg/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 µg/kg, IP) reduced sham intake, this reduction was not attenuated by MK-801 pre-treatment. 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-min gastric emptying was accelerated after MK-801 (3.9 + 0.2 ml) compared to saline vehicle (2.72 + 0.2 ml). CCK-8 (0.5 µg/kg, IP) reduced 10-min emptying to 1.36 + 0.3 ml. Pre-treatment with MK801 did not significantly attenuate CCK-8-induced reduction of gastric emptying (0.9 + 0.4 ml). This series of experiments demonstrate 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 following 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.
Key words: sham feeding, gastric emptying, MK-801.
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INTRODUCTION Meal size and meal termination are controlled by interplay between positive and negative feedback signals. Satiation signals, such as gastric distention, cholecystokinin (CCK) and the presence of nutrients in the small intestine limit the intake of food. Reduction of food intake by exogenous CCK depends upon activation of small unmyelinated vagal sensory fibers (12, 15). However, the neurotransmitter(s) released in the brain when these fibers are activated remains unknown. Several lines of evidence suggest that glutamate may be a vagal sensory neurotransmitter (16, 21, 26). A number of laboratories, including our own, have reported that MK-801 (dizolcipine), a non-competitive antagonist of N-methyl-D-aspartate (NMDA) receptor ion channels, increases feeding (1, 3-7, 13, 20-23, 26) as well as drinking (7, 24). MK-801 increases meal size initiated by an overnight food deprivation or the presentation of a highly palatable food (3), and has also been shown to increase intake of chow when administered just prior to dark onset in ad libitum fed rats (7). We have also shown that direct injection of MK801 into the dorsal vagal complex increases the size of deprivation-induced meals (21). Previous results suggest that blockade of NMDA receptors attenuates reduction of food intake by exogenous CCK (1). In the work reported here, we have compared the effect of NMDA receptor blockade on CCK-induced reduction of real feeding and sham feeding in order to determine whether increased food intake following NMDA receptor blockade is the direct result of interference with CCK mediated feedback signals. We also examined the effect of NMDA receptor blockade on inhibition of gastric emptying by CCK. We found that while NMDA receptor blockade reverses inhibition of real feeding by CCK, it fails to reverse inhibition of sham feeding or gastric emptying by CCK. These results suggest that MK-801 increases feeding by interfering with post-oral satiation signals that are modulated by CCK.
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METHODS Subjects: Adult (350-400 g) male Sprague-Dawley rats (Simonsen Laboratories, CA) were individually housed in hanging wire bottom cages in a temperature-controlled vivarium with ad libitum access to standard pelleted rodent chow and water except during experiments or overnight fasts as indicated below. The rats were maintained in a 12:12-h light-dark cycle (lights on at 0700) and were habituated to laboratory conditions for at least 5-7 days before surgery or the initiation of experiments. Drugs: MK-801 (dizolcipine, RBI, Natick, MA) and cholecystokinin octapeptide sulfate (CCK-8, (American Peptide, Inc., Sunnyvale, CA) were dissolved in physiological sterile saline and were administered intraperitoneally in a volume of 1.0 ml/kg body weight. Surgical Procedures: Gastric cannulation. Rats used in gastric emptying studies were implanted with stainless steel gastric cannulas, according to a modification of the procedure previously described by Yox and Ritter (25). Briefly, the animals were anesthetized with methoxyflurane (Metofane, Pitman-Moore, Mundelein, IL), and the flanged end of a stainless steel gastric cannula (13 mm long, 6 mm ID, 8 mm OD) was inserted through the ventral wall of the nonglandular portion of the stomach near the greater curvature. The cannula was secured with a purse-string suture, a piece of Marlex mesh was placed around it, and the nonflanged end of the cannula was externalized through an incision in the left paramedian abdominal wall. The cannula was kept closed with a stainless
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steel screw, except during experiments. A minimum of 2 weeks was allowed for recovery from surgery. Experiment 1: Effects of MK-801 on CCK-induced inhibition of solid and liquid food intake Solid food. Food deprived (17h) rats (n = 10) were injected with one of the following drug combinations: NaCl/NaCl, NaCl/MK-801, CCK/NaCl, CCK/MK-801. A pre-weighed amount of standard rodent chow was presented to the rats and food intake was recorded at 0.5, 1, 2, 4, and 6 h post injection taking into account spillage that was collected in a tray placed below the cage. MK-801 (100 µg/kg, IP), was administered 10 min prior to CCK-8 (2 or 4 µg/kg, IP) injection and 30 min prior to food presentation. CCK was administered 20 min prior to food. Each drug treatment was separated by a minimum of 48 hours and was bracketed by a NaCl/NaCl condition. Two separate studies were performed. In the first study, the dose of CCK used was 2 µg/kg whereas in the second one 4 µg/kg dose of CCK was administered. For each experiment, a minimum of two repetitions of each drug combination were conducted. Thus, each experimental datum represents the mean from at least two tests separated by a saline-only baseline tests. Liquid food. A separate group of rats (n = 8) were trained to drink a 15% sucrose solution (w/v) from a calibrated 25-ml glass burette. This group received the identical drug treatment combinations as described in Experiment 1, however, only the 2 µg/kg CCK dose was tested. Sucrose intake was measured every 5 minutes for 30 minutes in the place of measuring chow intake. Experiment 2: Effects of MK-801 on CCK-induced inhibition of gastric emptying. Gastric emptying after CCK. Rats (n = 9) were fitted with chronic gastric cannulae as described under Surgical Procedures. The rats were deprived of food, but not water, overnight for 17 h, before the start of gastric-emptying measurements. At the beginning of each gastric-emptying
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experiment, each rat was removed from its home cage, the gastric cannula was opened, and the stomach was gently washed with warm (37°C) tap water. A drainage tube was attached to the open cannula, and the rat was placed in a Plexiglas gastric-emptying cage, which has been described previously (25). The drainage tube exited through a longitudinal slot in the wire-mesh floor of the cage and rested in a graduated cylinder. After connection of the drainage tube and while the rats rested in the Plexiglas cages, the stomach was flushed twice with warm 0.9% NaCl, via the drainage tube, by means of a syringe attached to the drainage tube. The second saline wash contained phenol red (60 mg/l) to saturate gastric mucosal binding of phenol red and minimize loss of the dye due to adsorption during subsequent emptying measurements (8). Phenol red recovery after this procedure was 95-97% of the infused loads. After the final, dyecontaining wash, 30 min were allowed to drain any remaining wash solution from the stomach. Following this draining period, MK-801 (100 µg/kg) or NaCl was administered 15 min prior to a 5 ml 0.9%NaCl load. CCK (0.5 µg/kg) was given 10 min after MK-801 administration. The dose of CCK employed in this experiment was chosen based on previous studies examining the effects of CCK on gastric emptying (17). Rats received one of the following drug combinations: NaCl/NaCl, NaCl/MK-801, CCK/NaCl, CCK/MK-801. Five min after CCK or NaCl administration 5 ml of warm 0.9% NaCl, containing 0.006% phenol red, were instilled into the stomach via the drainage tube, and the tube was clamped. At the end of a 10-min emptying period, the clamp was removed, the volume remaining in the stomach was withdrawn, and the stomach was washed twice with saline and allowed to drain for another 30 min into a collection flask. Collected volume was measured, and the gastric contents were centrifuged at 10,000 rpm for 5 min to remove any particulate matter. A 1-ml sample from the centrifuged gastric contents was buffered with 24 ml of 0.014 M Na3PO4 · 12 H2O, and the spectrophotometric absorbance of
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each buffered sample was compared with that of a 1-ml buffered sample from the originally instilled phenol red solution to determine the volume of the original test load remaining in the stomach at the end of the 10-min emptying period. Each drug treatment was preceded and followed by gastric-emptying measurements after control injections of 0.9% NaCl. A minimum of two tests were conducted for each drug treatment, and all injections or infusions were separated by 48 h. Experiment 3. Effects of MK-801 on CCK-induced suppression of sham feeding. A separate group of rats (n = 7) equipped with chronic gastric cannulae were trained to sham feed 15% sucrose solution. Following overnight food deprivation, rats were removed from their cages, the stainless steel screw was removed from their gastric cannulas, and their stomachs were gently lavaged with warm tap water (37°C). After a drainage tube had been attached to their open cannula, the rats were placed in Plexiglas sham-feeding boxes. The rats then received one of the following drug combinations: NaCl/NaCl, NaCl/MK-801, CCK/NaCl, CCK/MK-801. MK-801 (100 µg/kg) or NaCl was administered 15 min prior to CCK (2 or 4 µg/kg) or NaCl. Calibrated glass burettes filled with 15% sucrose solution were presented immediately after CCK or NaCl injection and intake was recorded every 5 min for the ensuing 30 min. Two separate studies were performed. In the first study, the dose of CCK used was 2 µg/kg whereas in the second one 4 µg/kg dose of CCK was administered. For each experiment, a minimum of two tests were conducted for each drug combination. All drug administrations were separated by a period of 48 h during which no experimental manipulations occurred. Each drug combination was bracketed by a vehicle-only baseline combination. Statistical Analysis
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The cumulative food intake was analyzed with two-way repeated measures ANOVA with treatment and time as main factors using Sigma statistical software. The gastric emptying data were analyzed using one way repeated measure with treatment as the main factor. When a significant effect of treatment or an interaction was detected, Bonferroni tests were used in the post hoc analyses to isolate the effect. Intakes for all rats are expressed as means ± SE in milliliters for liquid food and in grams for solid food. RESULTS Effects of MK-801 on CCK-induced inhibition of solid and liquid food intake. Solid foods. Two-way repeated–measures ANOVA demonstrated significant main effects of drug combination [F(3, 199)=13.6, P
