Introduction

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The role of the endogenous opioid system in the mediation of ingestive behavior has been well established (for review, see Gosnell and Levine, 1996). Thus, direct microinjections of the endogenous opioid peptides -endorphin (Grandison and Guidotti, 1977) and dynorphin (Morley et al., 1982; Morley and Levine, 1983) stimulate feeding. Subsequently, selective opioid receptor agonists and antagonists were used to elucidate specific opioid receptor mechanisms modulating feeding. Hence, feeding induced by the endogenous opioid peptides has not been previously characterized in terms of receptor pharmacology. Recently, our laboratory (Silva et al., 2001) used general and selective (µ, , and ₁) opioid receptor antagonists as well as antisense oligodeoxynucleotide (AS ODN) probes directed against specific exons of the MOR-1, DOR-1, KOR-1, and KOR-3/ORL-1 opioid receptor genes to characterize feeding responses elicited by -endorphin, and found that the µ-opioid receptor was firmly implicated in the mediation of -endorphin-induced feeding based upon the magnitude of selective µ-opioid antagonism, and the effectiveness of AS ODN probes directed against either exons 1, 3, or 4 of the MOR-1 opioid receptor clone. The present study used an identical antagonist and AS ODN approach to characterize the pharmacology of the feeding response elicited by the endogenous opioid peptide dynorphin A_1-17.

Dynorphin is derived from post-translational cleavage of its precursor, preprodynorphin, initially isolated from porcine pituitary tissue (Goldstein et al., 1979). The identification of the nucleotide and amino acid sequences of preprodynorphin revealed C-terminal processing cleavage products, including dynorphin A_1-17, dynorphin A_1-13, and dynorphin A_1-8 (Goldstein et al., 1979; Kakidani et al., 1982). Dynorphin peptide fragments are widely distributed in the central nervous system, including structures involved in the mediation of ingestive responses such as the nucleus accumbens, basal ganglia, supraoptic and paraventricular hypothalamus, substantia nigra, and parabrachial nucleus (Khachaturian et al., 1982). Although dynorphin fragments display affinity for µ-, -, and -opioid receptors in traditional opioid brain homogenate binding studies (Huidobro-Toro et al., 1981; Wuster et al., 1981), the use of cloned opioid receptors indicates a 5- to 10-fold enhanced affinity of the dynorphin fragments for - relative to µ- and -opioid receptors (Mansour et al., 1995).

Feeding is elicited after ventricular administration of dynorphin A_1-17 and dynorphin A_1-13, which is blocked by general opioid antagonist pretreatment (Walker et al., 1980; Morley and Levine, 1981, 1983; Morley et al., 1982; Gosnell et al., 1986). Feeding is also elicited after intracerebral dynorphin administration into either the ventromedial and paraventricular hypothalamic nuclei, the ventral tegmental area, the median raphe, and the nucleus accumbens (for review, see Gosnell and Levine, 1996), but not the nucleus tractus solitarius (Kotz et al., 1997). In contrast, antibodies directed against dynorphin peptide fragments significantly reduce feeding elicited by electrical stimulation of the lateral hypothalamus (Carr and Bak, 1990). Dynorphin levels are increased by 2-deoxy-D-glucose-induced glucoprivation, chronic food restriction, streptozotocin-induced diabetes, and exposure to a palatable diet (Aravich et al., 1993; Berman et al., 1994, 1995, 1997; Welch et al., 1996). Although dynorphin-induced feeding has been postulated to act through interactions with -opioid receptors (for reviews, see Levine et al., 1985; Gosnell and Levine, 1996), only one selective antagonist study (Mann et al., 1988) demonstrated that dynorphin-induced feeding is unaffected by pretreatment with the selective µ₁-antagonist naloxonazine.

Therefore, the present study used two techniques to determine which opioid receptor subtypes participate in dynorphin A_1-17-induced feeding in rats: general and selective opioid antagonists, and AS ODN probes directed against opioid receptor genes. After determination of dose-response relationships for dynorphin-induced feeding, potential reductions were examined after pretreatment with a dose range (5-80 nmol) of general (Ntx), µ- (FNA), - (naltrindole), and - (NBNI) opioid antagonists. A second technique used AS ODN probes to establish the relationship of the cloned receptors to opioid actions using sequences that were complementary to regions of specific exons of mRNA to down-regulate opioid receptor proteins (Pasternak and Standifer, 1995), and provided converging evidence for feeding responses elicited by other opioid agonists (Leventhal et al., 1997, 1998a,b; Silva et al., 2001). The present study used AS ODN probes directed against specific exons of the MOR-1, DOR-1, KOR-1, and KOR-3/ORL-1 opioid receptor clones to analyze their effects upon dynorphin-induced feeding. Specificity of AS ODN effects was confirmed using an MS ODN probe that was identical to a particular effective AS ODN except that the order of three pairs of bases was reversed.

	Materials and Methods

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Subjects and Surgery. Adult male albino Sprague-Dawley rats (275-300 g; Charles River Laboratories, Inc., Wilmington, MA) were individually housed in suspended wire mesh cages and maintained on a 12-h light/dark cycle with rat chow pellets (Purina 5001 Rodent Diet, St. Lewis, MO) in food bins and water available ad libitum. All animals were pretreated with chlorpromazine (3 mg/kg i.p.) and were anesthetized with ketamine HCl (100 mg/kg i.m.). A stainless steel guide cannula (22-gauge; Plastics One, Roanoke, VA) was implanted stereotaxically (Kopf Instruments, Tujunga, CA) into the left lateral ventricle using the following coordinates: incisor bar (+5 mm), 0.5 mm anterior to the bregma suture, 1.3 mm lateral to the sagittal suture, and 3.6 mm from the top of the skull. Each cannula was secured to the skull by three anchor screws with dental acrylic. All animals were allowed at least 2 weeks to recover from stereotaxic surgery before behavioral testing began. After completion of behavioral testing, which took approximately 6 to 8 weeks for each animal, all rats were sacrificed with an overdose of anesthetic, and cannula placements were verified by visual inspection; all animals included in the data analyses had correct cannula placements in the lateral ventricle.

Dynorphin Dose-Response Curve. All behavioral testing was conducted in the home cage between 2 and 8 h after the onset of the light cycle to minimize circadian effects on food intake. Rats were adapted to at least 4 days of baseline testing to eliminate any novelty-induced feeding responses elicited by placement of the pellets on the floor of the cage. It should be noted that intake during this phase of the light cycle is minimal as reflected by the low control values. In this and all subsequent protocols, before any experimental conditions, the food bins were removed from each cage and replaced with preweighed food pellets. Each intake value was measured by the weight of the food pellets in grams and adjusted for spillage that was collected on paper towels placed below the wire mesh cage. After baseline measurements, a group of 15 cannulated rats was assessed for food intake after 2 h after microinjection of dynorphin A_1-17 (Peninsula Laboratories, Belmont, CA) at doses of 0, 5, 10, 20, and 50 µg in counterbalanced order at weekly intervals. All infusions were administered in a 5-µl volume of distilled water over 30 s through a stainless steel internal cannula (28-gauge; Plastics One) that extended 0.5 to 1.0 mm beyond the tip of the guide cannula, and which was connected to a Hamilton microsyringe by polyethylene tubing. After infusion, the internal cannula was removed and immediately replaced with a stainless steel dummy cannula (28-gauge; Plastics One) to prevent any effusion, and to ensure cannula patency between microinjection conditions.

General and Selective Opioid Antagonists, Dynorphin, and Food Intake. All antagonists were administered in 5-µl volumes of distilled water to guarantee solubility of the compounds. All 31 cannulated rats in the four antagonist studies were initially assessed for food intake 1, 2, and 4 h after vehicle and after dynorphin at a dose of 50 µg, which produced the most consistent feeding responses (see Results). The first subgroup of eight rats received the general opioid antagonist Ntx (Sigma-Aldrich, St. Louis, MO) at doses of either 1.89, 7.56, 15.12, or 30.24 µg (5-80 nmol) 1 h before dynorphin (50 µg) and were tested for food intake 1, 2, and 4 h after the second injection. The order of antagonist dose treatments in this and subsequent central antagonist protocols was counterbalanced across animals with a 1-week interval elapsing between treatments. The second subgroup of nine rats received the ₁-selective opioid receptor antagonist NBNI (Sigma/RBI, Natick, MA) at doses of either 3.65, 14.6, 29.2, or 58.4 µg (5-80 nmol) 1 h before dynorphin (50 µg) and were tested for food intake at 1, 2, and 4 h after the second injection. The third subgroup of seven rats received the -selective opioid receptor antagonist naltrindole (Sigma/RBI) at doses of either 20.4 or 40.8 µg (40-80 nmol) 1 h before dynorphin (50 µg) and were tested for food intake at 1, 2, and 4 h after the second injection. The fourth subgroup of six to eight rats received the µ-selective opioid receptor antagonist FNA (Sigma/RBI) at doses of either 2.45, 9.8, 19.6, or 39.2 µg (5-80 nmol) 24 h before dynorphin (50 µg) and were tested for food intake 1, 2, and 4 h after the second injection. The time interval between antagonist and agonist treatments reflected the respective peak and selective actions of the opioid antagonists (Sawynok et al., 1979; Portoghese et al., 1987, 1988; Arjune et al., 1990).

AS ODN Probes, Dynorphin, and Food Intake. As described previously, all groups of cannulated rats in the AS ODN studies were initially assessed for food intake after 1, 2, and 4 h after vehicle and after dynorphin at a dose of 50 µg, which produced significant feeding responses. All AS ODN probes were administered at 10-µg doses dissolved in 5-µl volumes of 0.9% normal saline based upon their previously determined effectiveness in feeding studies (Leventhal et al., 1997, 1998a,b; Silva et al., 2001) without producing nonspecific effects (for review, see Pasternak and Standifer, 1995). All phosphodiester oligodeoxynucleotides (Midland Certified Reagent, Midland, TX) were purified in our (G. W. Pasternak and G. C. Rossi) laboratory, and the identified locations of the AS ODN probes were based on the different opioid receptor gene sequences listed in GenBank (Table 1). The opioid AS ODN sequences directed against the individual exons of either the MOR-1, DOR-1, KOR-1, or KOR-3/ORL-1 opioid receptor genes used in the present study in rats are based upon the rat clone (for review, see Rossi and Pasternak, 1997). During each 6-day test phase, rats received microinjections of their particular AS ODN probes on days 1, 3, and 5 as previously described (Leventhal et al., 1997; Silva et al., 2001); this time course of treatment both down-regulates the synthesis of new receptors and permits turnover of existing receptors (for review, see Pasternak and Standifer, 1995). Rats were exposed to a maximum of two different AS ODN treatments with a minimal 2-week interval between AS ODN treatments. Subgroups of the 58 rats tested in this paradigm were assigned to the following conditions by matching increased food intake after dynorphin (50 µg) administration: AS ODN probes directed against either exons 1, 2, 3, or 4 of the MOR-1 gene (n = 7-8/condition); directed against either exons 1, 2, or 3 of the DOR-1 gene (n = 7-8/condition); directed against either exons 1, 2, or 3 of the KOR-1 gene (n = 7-8/condition); directed against either exons 1, 2, or 3 of the KOR-3/ORL-1 gene (n = 7-8/condition); or a MS ODN probe directed against exon 1 of the KOR-1 gene (n = 6), which differed from its corresponding AS ODN probe by the sequence reversal of three pairs of bases (Table 1). Twenty-four hours after the last AS or MS ODN treatment (day 6), all rats received dynorphin (50 µg), and food intake was assessed after 1, 2, and 4 h.

Statistics. To determine significant effects in each paradigm, separate one-way repeated measures analyses of variance were performed on cumulative food intakes after 1, 2, and 4 h. Tukey comparisons (p < 0.05) were used to determine individual significant agonist effects relative to vehicle treatment. Dunnett comparisons (p < 0.05) were used to determine individual significant antagonist or AS ODN effects relative to its corresponding dynorphin-induced feeding condition. Because antagonist and AS ODN effects upon dynorphin-induced intake failed to vary across the time course, data after 2 h are presented.

	Results

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Dynorphin-Induced Feeding Dose-Response Curve. Dynorphin produced significant dose-dependent increases in food intake with the three highest (10-50 µg), but not lowest (5 µg) doses significantly increasing intake (Table 2). Because the highest (50 µg) dose produced the consistent feeding responses that were comparable to those observed for morphine, morphine-6-glucuronide, and -endorphin (Leventhal et al., 1998b; Silva et al., 2001), this dose was used in all subsequent studies.

Ntx and Dynorphin-Induced Feeding. Dynorphin-induced feeding was significantly reduced only after pretreatment with the highest (80 nmol) Ntx dose after 2 h (Fig. 1A), suggesting some opioid mediation of dynorphin-induced feeding. However, pretreatment with none of the lower (5-40 nmol) Ntx doses was effective in significantly altering dynorphin-induced feeding.

View larger version (38K): [in this window] [in a new window]   Fig. 1.   Alterations (mean ± S.E.M.) in food intake (grams) after i.c.v. administration of either vehicle alone, dynorphin (50 µg) alone, or dynorphin after ventricular pretreatment with either the general opioid receptor antagonist Ntx at doses of either 5, 20, 40, or 80 nmol (A); the 1-opioid receptor antagonist NBNI at doses of either 5, 20, 40, or 80 nmol (B); the -opioid receptor antagonist naltrindole at doses of either 40 or 80 nmol (C); or the µ-opioid receptor antagonist FNA at doses of either 5, 20, 40, or 80 nmol (D). Significant differences in food intake were observed after all dynorphin treatment conditions relative to vehicle after 2 h in either naltrexone-treated [F(5,35) = 5.74, p < 0.0006], NBNI-treated [F(5,45) = 9.63, p < 0.0001], naltrindole-treated [F(3,21) = 7.71, p < 0.0016], or FNA-treated [F(5,35) = 7.92, p < 0.0001] groups. The asterisks (*) in this and subsequent figures indicate significant increases in dynorphin-induced feeding relative to corresponding vehicle control values (Tukey comparisons, p < 0.05). Please note that control intake was minimal during this testing period (2-8 h into the light cycle) in rats adapted to the testing paradigm such that the symbols often encompass the small S.E.M. values in this and subsequent protocols. The crosses (+) in this and subsequent figures indicate significant decreases in dynorphin-induced feeding after either antagonist or AS ODN pretreatment relative to corresponding dynorphin-induced feeding values alone (Dunnett comparisons, p < 0.05).

NBNI and Dynorphin-Induced Feeding. Dynorphin-induced feeding was significantly reduced after pretreatment with all NBNI doses after 2 h (Fig. 1B). The consistency of NBNI effects over this entire dose range strongly suggest ₁-opioid mediation of dynorphin-induced feeding.

Naltrindole and Dynorphin-Induced Feeding. Dynorphin-induced feeding was significantly reduced only after pretreatment with the highest (80 nmol), but not the lower (40 nmol) naltrindole dose after 2 h (Fig. 1C), suggesting some minimal -opioid mediation of dynorphin-induced feeding.

FNA and Dynorphin-Induced Feeding. Dynorphin-induced feeding was significantly reduced after pretreatment with the three higher (20-80 nmol), but not the lowest (5 nmol), FNA doses after 2 h (Fig. 1D). These data suggest µ-opioid mediation of dynorphin-induced feeding.

KOR-1 AS ODN Probes and Dynorphin-Induced Feeding. Dynorphin-induced feeding was significantly reduced by pretreatment with AS ODN probes directed against either exons 1 or 2 of the KOR-1 clone after 2 h (Fig. 2A). In contrast, an AS ODN probe directed against exon 3 of the KOR-1 clone failed to alter dynorphin-induced feeding. Importantly, administration of an MS ODN that differed from the AS ODN probe directed against exon 1 of the KOR-1 clone by the sequence reversal of only three pairs of bases also failed to alter dynorphin-induced feeding. The efficacy of AS ODN probes directed against exons 1 or 2 of the KOR-1 clone to significantly reduce dynorphin-induced feeding together with the ineffectiveness of the MS ODN probe suggests that the full expression of dynorphin-induced feeding is dependent upon the functional expression of exons 1 and 2 of the KOR-1 clone.

View larger version (45K): [in this window] [in a new window]   Fig. 2.   Alterations (mean ± S.E.M.) in food intake (g) after i.c.v. administration of either vehicle alone, dynorphin (50 µg) alone, or ventricular pretreatment over 3 days (days 1, 3, and 5) with AS or MS ODN probes with dynorphin administration occurring 24 h later (day 6). Significant differences in food intake were observed after either KOR-1 AS ODN treatment conditions [F(5,130) = 62.91, p < 0.0001] (A); KOR-3/ORL-1 AS ODN treatment conditions [F(4,76) = 38.71, p < 0.0001] (B); DOR-1 AS ODN treatment conditions [F(4,80) = 25.81, p < 0.0001] (C); or MOR-1 AS ODN treatment conditions [F(5,120) = 42.33, p < 0.0001] (D).

KOR-3/ORL-1 AS ODN Probes and Dynorphin-Induced Feeding. Pretreatment with AS ODN probes directed against either exons 1 or 2 of the KOR-3/ORL-1 clone significantly reduced dynorphin-induced feeding after 2 h (Fig. 2B). In contrast, dynorphin-induced feeding failed to be significantly affected by the AS ODN probe directed against exon 3 of the KOR-3/ORL-1 clone. These data suggest that the expression of dynorphin-induced ingestive response is dependent upon the functional expression of exons 1 and 2 of the KOR-3/ORL-1 clone.

DOR-1 AS ODN Probes and Dynorphin-Induced Feeding. In contrast to the significant effects of AS ODN probes directed against either the KOR-1 or KOR-3/ORL-1 clone, AS ODN probes directed against the DOR-1 clone failed to significantly alter dynorphin-induced feeding after 2 h (Fig. 2C), indicating that AS ODN probes targeting different exons of the DOR-1 clone are not involved in the mediation of dynorphin-induced feeding.

MOR-1 AS ODN Probes and Dynorphin-Induced Feeding. Dynorphin-induced feeding was significantly reduced after 2 h only by an AS ODN probe directed against exon 1 of the MOR-1 clone (Fig. 2D). AS ODN probes directed against either exons 2, 3, or 4 of the MOR-1 clone failed to alter dynorphin-induced feeding. These data suggest that the integrity of the MOR-1 clone plays a relatively minor role in the mediation of dynorphin-induced feeding.

	Discussion

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Increased food intake after dynorphin administration was significantly, dose-dependently, and differentially reduced by pretreatment with either general (Ntx), ₁- (NBNI), - (naltrindole), and µ-opioid (FNA) antagonists. These data reveal that NBNI significantly reduced dynorphin-induced feeding across the entire 5- to 80-nmol dose range, and FNA significantly reduced dynorphin-induced feeding after the three higher (20-80 nmol) doses. In contrast, only the highest (80-nmol) dose of either Ntx or naltrindole significantly reduced dynorphin-induced feeding. In addition, dynorphin-induced feeding was significantly reduced by AS ODN probes directed against either exons 1 and 2, but not 3 of the KOR-1 gene; exons 1 and 2, but not 3 of the KOR-3/ORL-1 gene; exon 1, but not 2 or 3 of the DOR-1 gene; and exon 1, but not 2, 3, or 4 of the MOR-1 gene. Furthermore, a control MS ODN probe that differed from the highly effective KOR-1 exon 1 AS ODN probe by the sequence reversal of only three pairs of bases was completely ineffective in altering dynorphin-induced feeding.

It has been strongly suggested that dynorphin-induced feeding acts through direct activation of the ₁-opioid receptor (for reviews, see Levine et al., 1985; Gosnell and Levine, 1996). Dynorphin displays 5- to 30-fold greater affinity for ₁-opioid receptors relative to µ- and -receptors (Mansour et al., 1995; Zhang et al., 1998), and its binding characteristics are very similar to the prototypical -agonist ethylketocyclazocine (Chavkin et al., 1982). The ability of dynorphin and other ₁-receptor ligands to increase feeding behavior is well established (Walker et al., 1980; Morley and Levine, 1981, 1983; Morley et al., 1982), and the hypothesis that it results from its direct interaction with the ₁-opioid receptor (for reviews, see Levine et al., 1985; Gosnell and Levine, 1996) was confirmed in the present study using two converging lines of evidence: a dose range of selective opioid receptor antagonists and highly selective AS ODN probes directed against specific exons of opioid receptor genes. The relatively high dose (80 nmol) of Ntx required to block dynorphin-induced feeding strongly parallels the low sensitivity to naloxone antagonism displayed by dynorphin as well as other -selective ligands relative to µ- and -selective agonists (Goldstein et al., 1979; Chavkin et al., 1982). The ability of ₁- and µ-opioid antagonists to significantly attenuate dynorphin-induced feeding is not unique to this agonist because both of these antagonists also significantly reduced feeding elicited by the ₁-selective agonist U50488H, and the µ-selective agonists [D-Ala²,N-Me-Phe⁴,Gly⁵-ol]-enkephalin and -endorphin (Leventhal et al., 1997; Levine et al., 1990, 1991; Silva et al., 2001). For example, the ability of NBNI at doses as low as 5 nmol to significantly reduce dynorphin-induced feeding compares favorably with this antagonist's ability to significantly reduce feeding elicited by U50488H (1 nmol; Levine et al., 1990). Yet, the ability of FNA at doses as low as 20 nmol to reduce dynorphin-induced feeding contrasts with its greater ability to effectively reduce feeding elicited by either [D-Ala²,N-Me-Phe⁴,Gly⁵-ol]-enkephalin (0.1-0.4 nmol; Levine et al., 1991; Leventhal et al., 1997) or -endorphin (0.5 nmol; Silva et al., 2001). The relative lack of efficacy observed for the -opioid antagonist naltrindole to reduce dynorphin-induced feeding was similar to its minimal effects upon -endorphin-induced feeding, and may reflect only a slight cross-activation of -receptors by dynorphin (Silva et al., 2001).

The results of the antagonist paradigms used in the present study are less compelling in light of the reduced sensitivity of dynorphin-induced feeding after the highest (80-nmol) doses of both the µ- (FNA) and  (naltrindole)-selective opioid antagonists. The cross-reactivity of naltrindole and FNA at multiple opioid receptors (for review, see Kieffer, 1995) may explain the lack of specificity of these antagonists at the highest dose observed in the present study. Alternatively, the ability of these selective antagonists to reduce dynorphin-induced feeding may reflect the activation of multiple opioid receptors by dynorphin. Indeed, in vitro binding studies have confirmed dynorphin binding to both µ- and -opioid receptor clones in addition to its observed -opioid receptor binding (Raynor et al., 1993; Mansour et al., 1995). Finally, the ability of µ- and -selective opioid receptor antagonists to reduce dynorphin-induced feeding suggests that this feeding response may be inherently dependent upon the activation of multiple opioid receptors downstream from the dynorphin site of action. Moreover, these results may reflect the essential contribution of multiple opioid receptors to the central control of feeding that is unrelated to mediation of dynorphin-induced feeding. However, a strong involvement of µ- and -opioid receptors in the mediation of dynorphin-induced feeding was not supported by the results from the AS ODN paradigm used in the present study.

The AS ODN studies provided compelling converging evidence supporting the hypothesis that -opioid receptors are primarily involved in the mediation of dynorphin-induced feeding. Dynorphin-induced feeding was eliminated by AS ODN probes directed against exons 1 and 2 of the KOR-1 gene. Such data are consistent with the ability of AS ODN probes directed against exon 1 of the KOR-1 gene to reduce feeding elicited by the ₁-selective opioid agonist U50488H, but not the morphine metabolite and µ-sensitive opioid agonist morphine-6-glucuronide (Leventhal et al., 1998b). This effect is also largely consistent with the specific and selective actions of AS ODN probes directed against the KOR-1 gene to reduce analgesic responses elicited by ₁-opioid agonists (Chien et al., 1994; Pasternak et al., 1999). Because AS ODN probes directed against each of the three exons of the KOR-1 gene were capable of reducing U50488H-induced analgesia (Pasternak et al., 1999), the inactivity of the exon 3 AS ODN probe argues against the possibility that the receptor responsible for dynorphin-induced feeding is completely encoded by the KOR-1 gene itself. Significantly, an equi-effective feeding response elicited by -endorphin was unaffected by AS ODN probes directed against exons 1 or 2 of the KOR-1 gene (Silva et al., 2001). The failure of the KOR-1 exon 1 MS ODN, which differed from the KOR-1 exon 1 AS ODN by the sequence reversal of only two pairs of bases, to alter dynorphin-induced feeding served as an important control measure to indicate the specificity of the AS ODN directed against exon 1 of the KOR-1 gene in altering dynorphin-induced feeding.

Interestingly, dynorphin-induced feeding was similarly reduced by AS ODN probes directed against exons 1 and 2, but not 3 of the KOR-3/ORL-1 gene. Although AS ODN probes directed against all three exons of the KOR-3/ORL-1 gene effectively eliminate feeding elicited by OFQ/N_1-17 (Leventhal et al., 1998a), an AS ODN probe directed against exon 1 of the KOR-3/ORL-1 gene failed to affect M6G-induced feeding (Leventhal et al., 1998a). Importantly, an equi-effective feeding response elicited by -endorphin was unaffected by AS ODN probes directed against exons 1 or 2 of the KOR-3/ORL-1 gene (Silva et al., 2001). Dynorphin and OFQ/N share significant primary structural similarities (Reinscheid et al., 1998). Specifically, the N-terminal domain known as the "message" domain of dynorphin is recognized by the KOR-3/ORL-1 receptor (Reinscheid et al., 1998). Moreover, as few as four point mutations in the amino acid sequence of the KOR-3/ORL-1 receptor results in the ability of this receptor to bind dynorphin A_1-17 with high affinity (Meng et al., 1996). Therefore, the sensitivity of dynorphin-induced feeding to reduction by AS ODN probes targeting exons 1 and 2 of the KOR-3/ORL-1 gene suggests that this receptor may play a significant role in the mediation of this ingestive response. These data thereby suggest that the full expression of dynorphin-induced feeding is dependent upon the integrity of both the KOR-1 and KOR-3/ORL-1 genes. This is somewhat surprising given the dissociation between ₁- and ₃-opioid agonists in feeding studies. Thus, although ₁-induced (U50488H) feeding is equieffectively reduced by both naltrexone and NBNI, ₃-induced (naloxone benzoylhydrazone) feeding is reduced by naltrexone, but not NBNI (Koch et al., 1992).

The roles of other opioid receptor subtypes in the mediation of dynorphin-induced feeding are less compelling in light of their AS ODN probe effects. The limited effectiveness of naltrindole (80 nmol) in reducing dynorphin-induced feeding is consistent with the inability of AS ODN probes directed against individual exons of the DOR-1 gene to alter this response. This is in contrast to the elimination of feeding elicited by the ₂ opioid agonist deltorphan by pretreatment with an AS ODN probe targeting exon 1 of the DOR-1 opioid receptor clone (Leventhal et al., 1998b). Although the highest doses (20-80 nmol) of the selective µ-opioid receptor antagonist FNA significantly reduced dynorphin-induced feeding, pretreatment with an AS ODN probe directed against only exon 1, but not exons 2, 3, or 4 of the MOR-1 gene reduced this effect. In contrast, AS ODN probes directed against either exons 1, 3, or 4 of the MOR-1 gene eliminated -endorphin-induced feeding (Silva et al., 2001).

Thus, the endogenous opioid peptide dynorphin A_1-17 stimulates food intake after microinjection into intracerebral sites historically implicated in ingestive behavior, including the ventromedial and paraventricular hypothalamic nuclei, the ventral tegmental area, the median raphe, and the nucleus accumbens (for review, see Gosnell and Levine, 1996), but not the nucleus tractus solitarius (Kotz et al., 1997). Furthermore, just as traditional pharmacological techniques have implicated a given agonist or antagonist in functional situations by exogenously administering that agent, other studies have implicated a particular endogenous ligand in a functional situation by observing changes in ligand levels after exposure to a behavioral event. Hence, quantification paradigms demonstrated that dynorphin concentrations in the central nervous system are increased by 2-deoxy-D-glucose-induced glucoprivation, chronic food restriction, streptozotocin-induced diabetes, and exposure to a palatable diet (Aravich et al., 1993; Berman et al., 1994, 1995, 1997; Welch et al., 1996). The wide distribution of dynorphin's ingestive effects in the central nervous system together with its activation under a wide array of ingestion-related situations strongly suggests that this opioid peptide is an important modulator of food intake. The present data now strongly suggest that the primary receptor site(s) of action by which dynorphin_1-17 stimulates food intake is the ₁- and also ₃-opioid receptor. As with the elucidation of receptor mediation of -endorphin-induced feeding in a companion study (Silva et al., 2001), the combined use of selective antagonists and additional modern molecular tools such as the AS ODN technique allow for the discovery of precise receptor mechanisms mediating feeding and other behavioral actions of the endogenous opioid peptide system.