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Am J Physiol Regul Integr Comp Physiol 285: R1046-R1054, 2003; doi:10.1152/ajpregu.00107.2003
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Peptides that Regulate Food Intake

Neuropeptide FF exerts pro- and anti-opioid actions in the parabrachial nucleus to modulate food intake

Danielle M. Nicklous and Kenny J. Simansky

Department of Pharmacology and Physiology, Drexel University College of Medicine, Philadelphia, Pennsylvania 19102

Submitted 3 March 2003 ; accepted in final form 28 July 2003

ABSTRACT

Neurons that synthesize the morphine modulatory peptide neuropeptide FF (NPFF; Phe-Leu-Phe-Gln-Pro-Gln-Arg-Phe-NH2) densely innervate the parabrachial nucleus (PBN), an area implicated in regulating food intake. We analyzed opioid-related actions of NPFF in feeding in adult male Sprague-Dawley rats. Unilateral infusion of 2 nmol/0.5 µl of the µ-opioid receptor agonist [D-Ala2,NMe-Phe4,glycinol5]enkephalin (DAMGO) into the lateral PBN increased 4-h food intake from 0.7 ± 0.1 to 3.3 ± 0.3 g. NPFF (1.25-5.0 nmol) prevented this hyperphagic µ-opioidergic action. In rats fed after 4-h deprivation (baseline = 12.3 ± 0.3 g/2 h), 5 nmol of NPFF did not alter and larger doses (10 and 20 nmol) actually increased food intake (+36, 54%). Twenty nanomoles also elevated intake of freely feeding rats (from 0.7 ± 0.1 to 5.1 ± 1.0 g/4 h). The opioid receptor blocker naloxone (10 nmol) antagonized this increase. These data reveal both pro- and anti-opioid actions of NPFF in the PBN to modulate feeding. The mechanisms for the opposite actions of low and high concentrations of this neuropeptide in parabrachial regulation of food intake remain to be determined.

feeding; hyperphagia; rats; FMRFamide; µ-opioid receptors

NEUROPEPTIDE FF (Phe-Leu-Phe-Gln-Pro-Gln-Arg-Phe-NH2) (NPFF) and the related octadecapeptide NPAF were first isolated from bovine brain (71) using antisera directed against the invertebrate tetrapeptide FMRFamide (Phe-Met-Arg-Phe-NH2) (49). Early studies focused on the actions of FMRFamide and NPFF-like peptides (NPFF-LP) in pain because of the common terminal dipeptide amide sequence with extended met-enkephalin (63). Intracerebroventricular administration of FMRFamide and NPFF-LP in rodents, for example, reduced the antinociceptive actions of morphine and lowered baseline thresholds for response in several models of pain (42, 63, 71). These data revealed apparently an anti-opioid role because intracerebroventricular injection of antisera against NPFF enhanced opioid-related analgesia and reversed tolerance to morphine (36, 40). Intrathecal administration of NPFF and FMRFamide, however, potentiated the analgesic effects of morphine and the {delta}-receptor agonist deltorphin. Furthermore, intrathecally these neuropeptides produced analgesia by themselves that was inhibited by opioid antagonists (22, 20). This pro-opioid activity occurred indirectly because NPFF and FMRF-amide displayed very poor affinity for µ-, {delta}-, and {kappa}-receptors (23, 51). Direct actions at two G protein-coupled receptors (NPFF1 and NPFF2) with high homology to the cholecystokinin/orexin/NPY receptor families have been demonstrated (9, 17). Thus mammalian NPFF-LP appear to function as physiological modulators of endogenous opioid activity and have been termed morphine-modulating peptides (see reviews by Refs. 47, 56). The direction and magnitude of the influence may depend on the relative stimulation of the receptor subtypes and the level of the neuraxis recruited for any particular response (e.g., Ref. 50).

NPFF-LP appear to be involved in processes other than pain, including cardiovascular regulation (29), novelty-induced stress (10), and reward (28). Given roles in sensory, autonomic, and behavioral functions, it is not surprising that these peptides have been implicated in regulating feeding. Intracerebroventricular FMRFamide reduced feeding elicited in mice by food deprivation, defeat in conspecific aggression, the {kappa}-receptor agonist U-50,488H, and morphine (32, 33, 34, 35). In rats, ~1 nmol of this tetrapeptide amide actually increased food intake in obese animals maintained on a palatable cafeteria diet, but not that of normophagic controls given free access to standard chow (54). Approximately 1 nmol icv of NPFF did reduce consumption of chow when normal rats were deprived of food for 24 h (45). Sunter et al. (62) reported that 3 and 10 nmol of NPFF decreased food intake in rats that were deprived overnight. The highest dose, however, occasionally produced brief periods of barrel rolling and immobility. Furthermore, both doses stimulated drinking. They attributed the hypophagic action of even the lowest dose of NPFF to competition from thirst rather than a selective action on feeding.

The anatomic sites for ingestive actions of NPFF-LP have not been explored. Immunocytochemical studies have revealed concentrations of NPFF-LP perikarya in the brain only in the nucleus tractus solitarius (NTS) and in medial hypothalamus between the dorsomedial, ventromedial, and periventricular nuclei (1, 30, 37, 38). The parabrachial nucleus (PBN) of the pons (especially the lateral PBN, LPBN) receives dense, bilateral projections of true NPFF from the solitary nucleus (38). The hypothalamus sends efferents to the PBN that also supply collaterals to the paraventricular nucleus of the hypothalamus; these neurons probably use a new, related NPFF-LP (NPVF) for neural communication (30, 42). Consistent with this pattern of innervation, the PBN expresses dense concentrations of NPFF receptors (2, 14, 21).

The PBN receives second-order oral and gastric afferents from the NTS and plays a significant role in integrating information subserving feeding (e.g., see Refs. 11, 27, 31, 41, 46, 58, 59, 61, 64). The PBN also expresses µ-, {kappa}-, and {delta}-opioid receptors, with µ-receptors being particularly dense (4, 43, 65, 67). We have evidence that infusing the µ-agonist [D-Ala2,N-Me-Phe4,glycinol5]enkephalin (DAMGO) into the LPBN elicits feeding in rats (60, 66). Furthermore, NPFF exerts electrophysiological actions via opioid mechanisms in the LPBN (13). Thus the LPBN would appear to be a sensible candidate for investigating functions of NPFF-LP in feeding.

The present study, therefore, analyzed the effects of infusing NPFF into the LPBN on feeding by rats. We assessed both the interactions of this neuropeptide with local, parabrachial administration of DAMGO and the ingestive responses to NPFF in otherwise untreated animals. We report that relatively low doses of NPFF prevent DAMGO-induced increases in feeding without affecting drinking. In contrast, higher doses of NPFF increase food intake by a mechanism that is blocked by the nonselective opioid receptor antagonist naloxone.

MATERIALS AND METHODS

Subjects

Adult male Sprague-Dawley rats (350-450 g) (Taconic Farms, Germantown, NY) were housed individually in suspended wire-mesh cages (43 cm length x 22 cm width x 18 cm height). The animal facility was maintained on a 12:12-h light-dark cycle, with lights on at 0600, at a temperature of 22-24°C. Water was freely available; standard pelleted chow (Purina, St. Louis, MO) was provided ad libitum except where noted. The procedures were performed in compliance with the "Guiding Principles for Research Involving Animals and Human Beings" of the American Physiological Society (3) and approved by the Institutional Animal Care and Use Committee (IACUC) of Drexel University.

Surgery

Rats were anesthetized with Equithesin (3.5 ml/kg ip), which was formulated to deliver ~36 mg/kg pentobarbital sodium and 160 mg/kg chloral hydrate. Rats were implanted unilaterally with a stainless steel 26-gauge guide cannula (Plastics One, Roanoke, VA) using the flat-skull technique with a stereotaxic instrument (Kopf Instruments, Tujunga, CA). The stereotaxic coordinates (4.8 mm below the skull surface, 1.8 mm lateral to midline, and 9.5 mm caudal to bregma) were located 1 mm above the intended infusion site in the lateral parabrachial nucleus (LPBN) (48). In accordance with requirements of the IACUC, buprenorphine hydrochloride (Sigma, St. Louis, MO) (0.2 mg/kg) was administered as a postoperative analgesic after recovery from the anesthesia on the day of surgery, twice the day after surgery, and once the following day. The IACUC approved one experiment without postoperative analgesia (noted below under Experimental Design). A stainless steel 33-gauge obturator, which ended flush with the tip of the guide cannula, remained in place except when infusions were made.

Immunohistochemistry

Three rats were anesthetized and perfused transcardially with 10% phosphate-buffered formalin (pH 7.4; Fischer, King of Prussia, PA) using a peristaltic pump (Cole Parmer Instrument, Vernon Hills, IL). The brains were removed, immersed in phosphate-buffered formalin for ~1 h, and then transferred to 0.1 M sodium phosphate buffer (PBS) containing 30% (wt/vol) sucrose for at least 24 h. The brain stems were blocked, frozen at -16°C, and 30-µm-thick sections (Leica cryostat model CM3050, Deerfield, IL) were collected in PBS. After a series of PBS washes, the sections were incubated first in PBS containing 10% normal goat serum (NGS) (Vector Laboratories, Burlingame, CA) and 0.3% Triton X-100 for 30 min at room temperature to reduce background staining. Sections were then incubated with the primary antibody, rabbit anti-NPFF polyclonal antibody (Chemicon International, Temecula, CA), diluted 1:5,000 in 4% NGS/PBS for 24 h at room temperature. As a control, sections were incubated in 4% NGS/PBS without the primary antibody. After three 10-min washes with PBS, sections were incubated with biotinylated goat anti-rabbit secondary antibody (Vector Laboratories) diluted 1:200 in 4% NGS/PBS for 1 h at room temperature. Sections were rinsed with PBS and exposed to an avidin/biotinylated enzyme complex (ABC) (Vectastain ABC kit; Vector Laboratories). After rinsing with PBS, staining was visualized using a 3,3'-diaminobenzidine (DAB) substrate kit (Vectastain DAB kit; Vector Laboratories; 3-5 min). Sections were rinsed and mounted on slides subbed with gelatin and chromium potassium sulfate dodecahydrate, dehydrated, and placed under coverslips with Per-mount. Sections were viewed under a Leitz Aristoplan (Wetzlar, Germany) microscope, and digital pictures were taken using a Leica DC-200 (Deerfield, IL) camera linked to Leica DC Viewer software.

An alternate set of sections through the parabrachial nucleus was used for double-labeling of NPFF and µ-opioid receptors. After a series of PBS washes, the sections were incubated first in PBS containing 10% normal donkey serum (NDS) (Vector Laboratories) and 0.3% Triton X-100 for 30 min at room temperature to reduce background staining. Sections were then incubated simultaneously with two primary antibodies for 24 h at room temperature (each diluted 1:5,000 in 4% NDS/PBS): rabbit anti-NPFF polyclonal antibody and guinea pig anti-µ-opioid receptor polyclonal antibody (both antibodies from Chemicon International). After three 10-min washes with PBS, brain sections were incubated simultaneously with two secondary antibodies for 1 h at room temperature (each diluted 1:200 in 4% NDS/PBS): donkey anti-rabbit IgG conjugated with rhodamine and donkey anti-guinea pig IgG conjugated with FITC (both secondaries from Jackson Immunoresearch Laboratories, West Grove, PA). Both secondary antibodies were cross-adsorbed by the manufacturer to ensure specificity for primary antibodies raised in rabbit or guinea pig, respectively. After three 10-min washes with PBS, sections were mounted onto glass slides and placed under coverslips with Vectashield mounting medium (Vector Laboratories). Fluorescent labeled sections were visualized with a fluorescence microscope (Leitz Aristoplan), and digital pictures were taken using a Leica DC-200 camera linked to Leica DC Viewer software.

Histological Analysis

At the conclusion of each experiment, the rats were anesthetized and perfused as for immunocytochemistry transcardially with 10% phosphate-buffered formalin (pH 7.4; Fischer) using a peristaltic pump (Cole Parmer Instrument). The brains were removed and frozen at -16°C, and 40-µm sections were taken using a Leica cryostat, model CM3050 (Deerfield, IL). The sections were stained with cresyl violet acetate (Sigma, St. Louis, MO) and projected onto templates modified from the atlas of Paxinos and Watson (48) using a camera lucida (Bausch and Lomb, Rochester, NY). Placements were localized from these projections.

Testing Procedure

All drugs were dissolved in 0.15 M sterile saline on the day of the experiment. They were infused into the LPBN as reported previously (59) using a stainless steel 33-gauge microinjector (Plastics One) that extended 1 mm below the end of the guide cannula. Infusions of 0.5-µl total volume were delivered over 90 s using a Harvard Apparatus model 975 infusion pump (South Natick, MA). Injectors were left in place for 30 s after infusion to minimize backflow. Food was provided immediately after each rat was returned to its home cage, and intake, corrected for spillage, was measured to 0.1-g precision at regular time intervals (as determined by each experiment). Experiments began when the baselines varied <10% on 3 successive days, typically 10-14 days after surgery. Injection of saline vehicle preceded each test day; no infusions were made on the day after a test. Therefore, drug treatments were assessed every 3rd day. As determined by ANOVA, baselines did not vary during the course of the experiments. Thus the mean of all vehicle pretest days for each rat was used as the baseline value for statistical analysis of the drug treatments. All data were analyzed using the SigmaStat v2.03 software program (SPSS, Chicago, IL).

Experimental Design

Inhibition of the hyperphagic action of DAMGO by NPFF. Ten rats were infused into the PBN with 0 (vehicle), 1.25, 2.5, and 5.0 nmol NPFF (Phe-Leu-Phe-Gln-Pro-Gln-Arg-Phe-NH2; F-8-F-NH2) (mol wt 1080); each dose of NPFF was followed 10 min later by 2.0 nmol DAMGO (mol wt 514) (both drugs from Bachem, King of Prussia, PA). During the course of this experiment, animals were allowed ad libitum access to food and water. Testing began at 1000 each day. Fresh preweighed pellets were provided, and food intake was measured 30, 120, and 240 min after the second injection. The dose of DAMGO (2.0 nmol) produced 80-90% of the maximal increase in food intake in previous work (e.g., see Refs. 60, 66). Each rat received the following drug combinations: vehicle/vehicle, vehicle/DAMGO, NPFF (all doses)/DAMGO, and NPFF (5 nmol)/vehicle. In the initial series of treatments, we tested the 5-nmol dose of NPFF in combination with DAMGO. As shown in RESULTS, 5 nmol of NPFF eliminated the action of DAMGO to increase feeding. To estimate a dose-response relationship, we conducted subsequent experiments using 2.5 nmol and then 1.25 nmol of NPFF plus DAMGO. These data were systematic, and we did not replicate the design with a randomized or counterbalanced design. An additional study on selectivity of action of NPFF (below) did replicate the fundamental finding that NPFF inhibits DAMGO-induced feeding. The data were analyzed as specified in RESULTS by one-way repeated-measures ANOVAs followed by Student-Newman-Keuls tests for pairwise comparisons of means. An {alpha}-level of P < 0.05 was taken as the threshold for statistical significance.

Selectivity of actions of NPFF and DAMGO on food and water intake. A separate group of five rats was prepared with lateral parabrachial implants. Postoperative analgesia was not administered (cf. Ref. 62). The rats were tested with 2.0 nmol DAMGO, 5.0 nmol NPFF, and their combination as above, except that water was monitored in addition to food for the 4-h test. We determined baselines after a double infusion of vehicle on the day preceding each of the tests with the peptides. Statistical analyses used the average of the three baselines for each rat.

We assessed the effect of 2.0 nmol of DAMGO on consumption of water by the same rats when food was not available for the 4 h after infusion.

Dose response for the hyperphagic action of NPFF during scheduled feeding. Twelve experimentally naive rats were implanted unilaterally with guide cannulas into the LPBN. Five to seven days after surgery, animals were adapted to a feeding schedule in which they received ~27 g of chow daily (41, 59). This amount was equal to 95-100% of the average daily intake of the rats during 24-h free feeding and produced reliable, stable food intakes for the measurement period. Beginning at 1400 daily, rats were infused unilaterally into the PBN with 5.0, 10.0, or 20.0 nmol of NPFF. Food was given immediately after the injection, and food intake was measured for the next 30 and 120 min. Any remaining pellets were removed at 1000 the next day.

Dose response for the hyperphagic action of NPFF during free feeding. Once we established the effects of NPFF on scheduled feeding, the same animals (n = 12) were adapted to a free-feeding schedule (ad libitum access) and retested with 10 and 20 nmol NPFF. Testing began at 1000 daily. Food intake was measured 30, 120, and 240 min after injection. Data were analyzed by one-way and two-way, repeated-measures ANOVA followed by Student-Newman-Keuls tests for pairwise comparisons of means.

Inhibition of the hyperphagic action of NPFF by naloxone. The animals for this experiment were the same animals used to determine the effects of NPFF on scheduled and free feeding. To test whether 10 nmol of naloxone hydrochloride (10 nmol; mol wt 364; Sigma, St. Louis, MO) would antagonize the hyperphagia produced by NPFF during free feeding, we infused six animals with vehicle followed by NPFF (20 nmol) and the other six animals with naloxone (10 nmol) followed by NPFF (20 nmol). This dose of naloxone blocked completely the hyperphagic action of DAMGO (2 nmol) in satiated animals (60, 66). Testing began at 1000 daily. Food was given immediately after the second injection; intake was measured 30, 120, and 240 min postinjection. All rats were tested with vehicle + NPFF 1 wk before this test to ensure that the two groups were equally responsive to NPFF. Data were analyzed by one-way repeated-measures ANOVA followed by Student-Newman-Keuls tests for pairwise comparisons of means.

RESULTS

Immunocytochemical and Histological Analysis

As reported previously (30, 37, 38), neuronal processes staining for NPFF-like immunoreactivity innervated the LPBN (Fig. 1). Positive fibers were particularly dense in the region of the external lateral subnucleus. The distribution of NPFF overlapped with that for µ-opioid receptors (cf. Ref. 12). Infusion loci were concentrated particularly at this level within the LPBN between 9.16 and 9.30 mm caudal to bregma [referred to the atlas of Paxinos and Watson (48)]. Overall, sites ranged from the coronal level at which the motor nucleus of the trigeminal nerve appears rostrally, to the level of the accessory abducens nucleus caudally. One of the implants resulted in infusions much more rostrally and medial to the parabrachial complex at the ventrolateral edge of the mesencephalic trigeminal nucleus. This rat was excluded from the study because it did not eat more in response to DAMGO. One rat lost its cannula during the experiment and was excluded from all analyses.



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  		Fig. 1. Neuropeptide FF (NPFF)-like and µ-opioid receptor-like immunoreactivity at the pontine level where cannula implants were concentrated in the lateral parabrachial nucleus (LPBN). Filled circles in schematic [top left; modified after the atlas of Paxinos and Watson (48)] indicate typical placements of infusions. The filled gray structure represents the superior cerebellar peduncle (scp in other panels), and the area filled by hatching is the external lateral subnucleus (LPBE). The LPBN includes this area and the surrounding region lateral and dorsal to the scp. DRc, dorsal raphe nucleus, caudal aspect; LC, locus ceruleus; Mo5, motor nucleus of the trigeminal nerve; MPBE, external medial subnucleus. Top right panel shows dense NPFF-like staining revealed by 3,3'-diaminobenzidine (DAB) chromogen and centered on the external lateral subnucleus. Double-labeling for immunofluorescence for NPFF and µ-opioid receptors (µ-OR) in the boxed area is shown in middle panels. Note broader distribution of receptors than peptidergic processes but considerable overlap along the mid- and ventrolateral aspect of the scp. This region includes the external lateral and the central lateral subnuclei. Overlay in bottom panel shows common distribution of punctate labeling for NPFF and µ receptors in the external lateral subnucleus.

 

Parabrachial Infusion of NPFF Inhibits the Hyperphagic Actions of DAMGO

Parabrachial infusion of DAMGO (2 nmol) increased 4-h food intake to 3.3 ± 0.3 g from a baseline of 0.7 ± 0.1 g. Pretreatment with NPFF reduced this hyperphagia in a dose-related manner (ED50 {cong} 1.8 nmol) with the highest dose returning intake to baseline levels, F(3,27) = 20.71, P < 0.01 (Fig. 2). Analysis of the intakes within the sequential time intervals of the tests (Fig. 3) revealed that intakes differed as a function of treatment, F(4,36) = 22.80, P < 0.01; interval, F(2,18) = 15.04, P < 0.01; and their interaction, F(8,72) = 3.88, P < 0.01. Note that DAMGO (0 nmol pretreatment in Fig. 3) increased eating above baseline (vehicle) in the two latter periods and that the inhibitory effect of NPFF persisted throughout the test. The highest dose of NPFF did not change food intake compared with baseline in these rats (4-h intake for 5 nmol NPFF + vehicle = 0.5 ± 0.1 g). Furthermore, although we did not make formal observations, the rats appeared to display normal periprandial behaviors during the tests.



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  		Fig. 2. NPFF pretreatment inhibits the hyperphagic action of [D-Ala2,N-Me-Phe4,glycinol5]enkephalin (DAMGO). Ten rats were infused into the LPBN with NPFF (1.25-5.0 nmol) or vehicle (0-nmol dose) followed 10 min later with 2.0 nmol DAMGO. Baseline (0.7 ± 0.1 g) is indicated by horizontal line with single break and represents the 4-h food intake after 2 infusions of vehicle. DAMGO (0-nmol pretreatment) increased food intake above baseline (P < 0.01). Difference from 0-nmol pretreatment + DAMGO: *P < 0.05, **P < 0.01, Student-Newman-Keuls test.

 


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  		Fig. 3. Food intakes (means ± SE) within the 3 measurement intervals during the 4-h test described in Fig. 2. Vehicle represents condition in which vehicle was administered twice into the LPBN (baseline). The other bars are data for conditions in which rats received 2.0 nmol DAMGO after pretreatment with 0-5.0 nmol NPFF. Difference from 0 nmol NPFF + DAMGO condition (*P < 0.05; **P < 0.01) or vehicle + vehicle condition ({dagger}P < 0.05; {ddagger}P < 0.01); Student-Newman-Keuls test.

 

Comparison of Effects of NPFF on Food and Water Intakes

DAMGO increased 4-h food intake from a baseline of 1.4 ± 0.1 g (after vehicle + vehicle) to 5.3 ± 0.7 g, P < 0.01. NPFF prevented this increase (NPFF + DAMGO, 1.0 ± 0.3 g) but did not alter baseline by itself (1.2 ± 0.4 g). After DAMGO, rats increased their consumption of water (5.8 ± 1.9 ml) from a baseline of 3.1 ± 0.5 ml. This increase was quite variable, however, and did not reach statistical significance, F(3,12) = 2.024, P > 0.10. After NPFF, rats drank water at baseline levels (NPFF + vehicle, 3.2 ± 0.5 ml; NPFF + DAMGO, 2.4 ± 0.5 ml). When rats were infused with DAMGO but not given access to food, they drank 3.4 ± 1.0 ml of water, which was identical to 4-h intake after vehicle (3.4 ± 0.4 ml).

NPFF-Induced Hyperphagia During Scheduled Feeding

Although by itself, 5 nmol of NPFF did not influence eating, the paradigm used above produced small baselines that would be insensitive to inhibitory actions of drugs. Accordingly, we prepared another group of rats that were maintained on a feeding regimen that produced high, stable baselines (2-h intake after vehicle = 12.3 ± 0.3 g). NPFF surprisingly increased food intake in a dose-related manner, F(3,33) = 9.80, P < 0.01. The 10 nmol (17.6 ± 1.4 g, P < 0.01) and 20 nmol (19.8 ± 1.6 g, P < 0.01) but not 5 nmol (13.2 ± 0.9 g) doses of NPFF increased total consumption of pellets for the 2-h test (not depicted). Figure 4 shows the amounts of chow eaten during the initial 30-min and subsequent 90-min periods after administration of the octapeptide. The elevated intakes occurred only after infusion of the two higher doses and solely within the second measurement period [dose x interval interaction, F(3,33) = 15.75, P < 0.01].



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  		Fig. 4. Parabrachial infusion of NPFF increases food intake (means ± SE) by rats maintained daily on a regimen in which they were deprived of food for 4 h during the light period. Difference from 0 nmol NPFF (vehicle): **P < 0.01; Student-Newman-Keuls test.

 

NPFF-Induced Hyperphagia in Freely Feeding Rats

With the finding that NPFF increased scheduled food intake, we tested the two highest doses in the same rats after they were adapted to ad libitum access to food (Fig. 5). NPFF increased food intake, F(2,22) = 13.53, P < 0.01, more food was eaten in the longer, later two intervals than in the initial interval (both P values < 0.05), and there was an interaction between dose and interval, F(4,44) = 3.00, P < 0.05. Only the 20-nmol dose of NPFF increased food intake, and this hyperphagic action occurred during the later two intervals.



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  		Fig. 5. Parabrachial infusion of NPFF increases food intake (means ± SE) by rats given free access to food. Difference from 0 nmol NPFF (vehicle): **P < 0.01; Student-Newman-Keuls test.

 

Inhibition of NPFF-Induced Feeding by Opioid Receptor Blockade

Pretreatment with 10 nmol of the opioid receptor antagonist naloxone reduced greatly the hyperphagic action of NPFF in freely feeding rats (Fig. 6).



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  		Fig. 6. Parabrachial infusion of naloxone (Nal; 10 nmol) inhibits feeding elicited by NPFF (20 nmol) in rats given free access to food. Bars represent food intakes (means ± SE) after infusion of either vehicle (Veh) followed by NPFF (n = 6) or naloxone followed by NPFF (n = 6). Responses to vehicle + NPFF were determined 1 wk before this test. The Vehicle/NPFF group ate 5.3 ± 1.9 g and the naloxone/NPFF group ate 4.8 ± 0.7 g during this pretest. Difference between groups: **P < 0.01; Student's t-test, 2-tailed.

 

DISCUSSION

These are the first data demonstrating ingestive actions of NPFF after administration into a discrete region of the brain. The results establish that this peptide can oppose orexigenic effects of µ-opioid receptor stimulation within the LPBN. The dose of NPFF that prevented DAMGO-induced feeding did not alter baseline water intake. This finding differed from the dipsogenic effect reported previously for NPFF administered intracerebroventricularly (62). Indeed, parabrachial infusion of NPFF reduced drinking produced by DAMGO in some rats. That drinking was clearly prandial, however, and NPFF merely returned water intake to basal levels in parallel with food. We infer that NPFF acted primarily on the feeding-related circuitry of the PBN to inhibit the hyperphagic response to DAMGO.

In contrast to the anti-opioid action of NPFF described above, higher doses increased food intake. This hyperphagia occurred in rats that were maintained either on an ad libitum schedule that engendered very low baselines or on a regimen with brief deprivation that led to much higher baselines. Naloxone decreased the heightened feeding, thereby implicating opioid mechanisms in this effect of NPFF.

The actions of low doses of NPFF to decrease and higher doses to mimic responses to opioids might suggest that this neuropeptide acts as a partial opioid agonist with similar, high affinity for the µ-opioid receptor compared with DAMGO. Nonetheless, NPFF displays little affinity for any of the subtypes of opioid receptors (23, 51). Thus NPFF acted presumably via its own receptors to physiologically antagonize DAMGO. In hippocampal slices, NPFF reduced the inhibitory actions of morphine on interneurons within the CA1 region although the octapeptide had no effect by itself (44). These investigators suggested that NPFF might inhibit K+ channels to antagonize opioid-mediated hyperpolarization (24). It is relevant also that an NPFF analog reversed the actions of DAMGO and nociceptin to inhibit calcium conductance in spinal ganglion cells (52) and in acutely dissociated neurons of dorsal raphe nucleus (57). These NPFFergic effects occurred at concentrations that did not themselves alter calcium conductance. Thus candidate cellular mechanisms for the present findings of anti-opioid actions in feeding exist for study within the PBN.

As demonstrated analogously in tests of nociception (see Introduction), NPFF mimicked hyperphagic effects of opioid stimulation in our study. The onset of feeding was delayed, as it was for DAMGO. The relatively long latency for hyperphagic responses to µ-opioid stimulation has been reported previously for infusions of DAMGO into the NTS (39) and ventral striatum, including the nucleus accumbens (7). Rats are initially hyperactive after infusion of DAMGO into the PBN, and this moderate increase in exploratory-type behavior continues into the period when feeding commences (60, 66). Thus the delayed feeding after DAMGO (and probably NPFF) cannot be explained by sedation or by response competition from other motor behaviors. One possibility is that the retardation in feeding is due to the kinetics of second messenger or other cellular mediating mechanisms [see discussion by Bakshi and Kelley (7)], although this has not been tested. Another is that µ-opioidergic stimulation must recruit second-order neuronal pathways to elicit feeding. The distribution of µ-sensitive sites from medulla to forebrain questions whether a common serial pathway is engaged by all of these disparate loci. Rats eat palatable food quickly after infusions of the benzodiazepine midazolam into the LPBN (26, 61). Thus the latency to eat after such treatments may be determined by the tone of neurochemical systems with which drugs interact. That tone is influenced by the stimulus properties of the food and other behavioral conditions of the experiment.

Several mechanisms may contribute to NPFF-induced feeding. In spinal ganglion cells, NPFF-LP produced actions similar to those of µ- and {delta}-agonists by reducing the rise in internal calcium observed during depolarization (55). Additionally, a peptidase-resistant analog of NPFF produced spinal analgesia that was antagonized by the {delta}-antagonist naltrindole (68). Furthermore, intrathecal administration of an NPFF analog increased the outflow of met-enkephalin-like peptide(s) from spinal cord (8). Moreover, NPFF modulated excitatory transmission in the PBN in the same manner as the {delta}-agonist deltorphin, and a {delta}-antagonist blocked this synaptic action (13). Naloxone inhibited the hyperphagic effect of NPFF in our study, but this is a relatively nonselective opioid antagonist (19). It is possible, therefore, that NPFF released parabrachial enkephalin, which then acted at µ- and/or {delta}-receptors to elicit feeding (see also Ref. 13). One point we did not explore fully was whether background opioid tone influences the direction of effect of NPFF. Specifically, it remains to be determined whether the high doses of NPFF that by themselves produced feeding would reduce the ability of DAMGO to elevate intake. Thus, in vivo and in vitro, the relative concentrations of NPFF for anti-opioid cellular, opioid mimicking cellular, and opioid releasing actions remain to be determined in the presence and absence of heightened opioid tone. It is possible that different NPFF receptor subtypes mediate opposite influences on feeding, although recent data suggest that the PBN may express only NPFF2 (21). This question remains difficult to address at present because molecular and especially pharmacological tools to dissect NPFFergic mechanisms are early in development.

The neurotransmitter/neuromodulator systems that contribute to parabrachial regulation of feeding are emerging. Local administration of benzodiazepines, for example, increased feeding by enhancing the positive hedonic qualities of tastants (61). Thus GABAA receptors within this locus apparently mediate orexigenic activity. In our laboratory, activating serotonergic 5-HT1B receptors in the LPBN reduced food intake (41, 59). Certainly, interactions between these and opioidergic systems within the LPBN to modulate ingestion are possible. Ample evidence exists for analogous interplay between opioids and other neurotransmitters such as excitatory amino acids within the nucleus accumbens (16) or GABA within the accumbens and ventral tegmental area (15, 72). Nonetheless, we believe that the interaction of NPFF with µ-opioid receptors in feeding represents a special case of a more general opioid modulatory role for this peptide in the central nervous system. This assertion is based on the history of the interactions of NPFF-like peptides with opioids at several levels of the neuraxis and the specialized mechanisms proposed as the basis for those interactions.

The present results add opioidergic mechanisms and their modulation by NPFF-LP in the PBN to the list of neurotransmitters subserving parabrachial regulation of autonomic function and ingestion. These new findings complement the stimulatory role for µ-opioid receptors in the NTS in feeding (18, 39) and the anatomic evidence for a projection by NPFFergic neurons from the NTS to the PBN (30, 38). Evidence exists that µ-opioid receptors are expressed on somatodendritic elements of the PBN, including the dendrites of the external lateral subnucleus (12). The codistribution of NPFF-positive processes and µ-opioid receptors shown in Fig. 1 suggests either that NPFFergic axons or terminals express the receptor antigen and/or that the two antigens lie in close apposition on separate neurons. Their precise ultrastructural localization and spatial relationship(s) in the PBN have not been analyzed in this study or elsewhere in the literature.

The PBN receives second-order afferents from multiple sensory systems, including pathways that convey taste and gastrointestinal information (25). The lateral subregion emphasized in this paper encodes each of these types of stimuli (69, 70) and contains some neurons that integrate convergent input from both (e.g., 5, 6, 31). The possibility exists that opioid-NPFF interactions are involved in modulating gustatory and viscerosensory processing. Overall, the neurochemical organization of the circuitry, the cellular interactions of the various receptors, and the influence of taste and visceral feedback on activity of the different mechanisms remain to be determined.

DISCLOSURES

Research grants from the National Institute of Mental Health (MH-41987) and the National Institute of Diabetes and Digestive and Kidney Diseases (DK-58669) to K. J. Simansky supported this work.

ACKNOWLEDGMENTS

We thank Dr. V. J. Aloyo for helpful comments on this work and the manuscript.

FOOTNOTES  

Address for reprint requests and other correspondence: K. J. Simansky, Dept. of Pharmacology and Physiology, Drexel Univ. College of Medicine, Mailstop 488, 245 N. 15th St., Philadelphia, PA 19102-1192 (E-mail: simansky{at}drexel.edu).

FOOTNOTES

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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