Introduction

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In addition to the ability of endogenous opioid peptides and peptide analogues to stimulate food intake (see reviews by Gosnell and Levine, 1996; Morley et al., 1983), morphine and other opiates such as heroin, butorphanol, codeine and levorphanol also produce a robust feeding response (e.g., Levine and Morley, 1983; Levine et al., 1994; Sanger and McCarthy, 1980; Thornhill et al., 1976). Morphine is rapidly metabolized and glucuronidated at both the three and six positions (Jaffe and Martin, 1985). Although M6G labels mu receptors with an affinity slightly less than morphine in binding assays (Paul et al., 1989), it is 100-fold more potent (Paul et al., 1989) centrally on both thermal (Abbott and Palmour, 1988; Pasternak et al., 1987; Shimomura et al., 1971; Sullivan et al., 1989) and visceral (Frances et al., 1992) nociceptive tests than morphine. To investigate whether M6G, as with its parent compound, morphine, produces long-acting (4 hr) ingestive effects, the first goal of our study was to determine whether central administration of M6G dose-dependently increased spontaneous food intake in rats.

Selective opioid antagonists directed against mu (FNA), delta₁ (DALCE), delta₂ (NTII) and kappa₁ (NBNI) receptor subtypes have been used to assess opioid receptor subtype involvement of opioid agonist-induced feeding, including hyperphagia induced by selective mu opioid agonists (see review by Gosnell and Levine, 1996). Using this approach, it appears that feeding stimulated by selective opioid agonists may involve more than one opioid receptor subtype. For instance, hyperphagia elicited by the selective mu opioid agonist DAMGO is blocked by pretreatment with either FNA or NBNI (Levine et al., 1990, 1991). Therefore, our second goal was to determine whether M6G-induced hyperphagia was specifically mediated by mu opioid receptors by: 1) pretreating animals with equimolar doses of either mu (FNA), delta₁ (DALCE), delta₂ (NTII) or kappa₁ (NBNI) opioid antagonists, 2) examining dose-response effects of FNA and 3) examining FNA effects on food intake following vehicle treatment.

The cloning of the major opioid receptor classes (see review by Uhl et al., 1994), led to the development of AS ODN techniques to elucidate functional roles of opioid receptors by correlating the molecular biology of the cloned receptors to opioid actions in vivo (see review by Pasternak and Standifer, 1995). This approach has been especially fruitful in distinguishing M6G from morphine in analgesic assays (Rossi et al., 1995a, 1995b, 1997a). Detailed mapping studies of the four exons of the MOR-1 clone revealed that AS ODN probes targeted against either exons 1 or 4 of the MOR-1 clone blocked both DAMGO-induced and morphine-induced analgesia. In contrast, AS ODN probes targeted against exons 2 or 3 of the MOR-1 clone were ineffective. An opposite pattern was observed for M6G-induced analgesia in that AS ODNs probes targeted against either exons 2 or 3 of the MOR-1 clone were effective, although AS ODNs directed against exons 1 or 4 were ineffective. These behavioral observations are supported by biochemical data suggesting the existence of a novel M6G receptor resulting from alternative splice variants of the MOR-1 clone (Brown et al., 1997a). Further evidence for the existence of a novel M6G receptor is supported by studies using mu receptor-deficient CXBK mice, morphine-tolerant animals and transgenic mice with disruption of the MOR-1 gene (Brown et al., 1997a; Rossi et al., 1996; Schuller et al., 1997).

The AS ODN technique has been used to investigate spontaneous and opioid-induced feeding. AS ODNs directed against each of the four exons of the MOR-1 clone significantly reduced spontaneous food intake and body weight in rats (Leventhal et al., 1996). In contrast, mu opioid agonist-induced feeding displayed the identical profile of AS ODN effects as observed in analgesic studies. Specifically, hyperphagia elicited by DAMGO was significantly reduced by AS ODN targeted against either exons 1 or 4, but not exons 2 or 3, of the MOR-1 clone, implying that both mu agonist behaviors may be mediated by the same receptor (Leventhal et al., 1997). Therefore, our third and final goal was to explore the effects of AS ODN probes targeted against regions of the MOR-1, DOR-1, KOR-1 and KOR-3/ORL1 clones on M6G-induced hyperphagia. It was expected that M6G-induced hyperphagia, like M6G-induced analgesia, would be reduced by AS ODN probes directed against either exons 2 or 3, but not exons 1 or 4 of the MOR-1 clone. Therefore, three important controls are included. First, an MS ODN, based upon one of the probes (exon 2) that effectively blocks M6G-induced hyperphagia, but with nucleotide bases changed in three positions, was employed. Second, although morphine and DAMGO had similar AS ODN profiles in analgesic assays, it is important to confirm that AS ODN effects on morphine-induced hyperphagia are similar to that observed for DAMGO-induced hyperphagia (Leventhal et al., 1997), and distinct from the hyperphagic responses of its active metabolite, M6G. Third, it is necessary to confirm activity of AS ODN probes targeting DOR-1, KOR-1 and KOR-3/ORL1 clones if such probes fail to alter M6G-induced hyperphagia. Therefore, these probes were tested against their respective selective agonists in ingestive assays as well: DOR-1 (Delt II), KOR-1 (U50488H) and KOR-3/ORL1 (nociceptin).

	Materials and Methods

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Subjects. Male albino Sprague-Dawley rats (90-120 days old, Charles River Laboratories, Kingston, NY) were housed individually in wire mesh cages and maintained on a 12-hr light: 12-hr dark cycle with water and rat food available ad libitum. Each rat was pretreated with chlorpromazine (3 mg/kg, i.p.) and anesthetized with Ketamine HCl (120 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. Cannulas were secured to the skull by three anchor screws with dental acrylic. All animals were allowed at least 2 wk to recover from stereotaxic surgery before behavioral testing began. Rats weighed between 275 and 300 g before surgery, and weighed 400 to 550 g after completion of testing. After completion of behavioral testing, all animals were killed with an overdose of anesthetic (Ketamine HCl, 300 mg/kg, i.m.), and cannula placements were verified visually.

Opioid agonists, AS ODNs and opioid antagonists. The opioid agonists, M6G (Research Technology Branch, NIDA, Rockville, MD), morphine (Pennick Laboratories), nociceptin (synthesized by G.W.P.), Delt II (Peninsula Laboratories) and U50488H (Upjohn Laboratories), were each dissolved in 0.9% normal saline. All phosphodiester oligodeoxynucleotides (Midland Certified Reagent Company, Midland, TX) were dissolved in 0.9% normal saline at a concentration of 5 µg/µl and purified in our (G.W.P.) laboratory. Table 1 summarizes the sequences of: 1) the AS ODNs (19-22 bases long) that were directed against each of the four exons of the MOR-1 clone, 2) an MS ODN directed against exon 2 of the MOR-1 clone in which three bases from the antisense sequence had been changed and 3) AS ODNs directed against specified regions of the DOR-1, KOR-1 and KOR-3/ORL1 clones. These sequences were chosen because of their previously demonstrated effectiveness in specifically and selectively reducing analgesia induced by their respective opioid receptor subtype agonists (Chien et al., 1994; Pan et al., 1995; Rossi et al., 1994, 1995a, 1995b, 1997a, 1997b). Analysis of the GenBank revealed that each of the AS ODN sequences were specific to the region targeted for the MOR-1, DOR-1, KOR-1 and KOR-3/ORL1 clones, and are not present in other opioid receptor cDNAs. The opioid antagonists, FNA (Research Biochemicals Intl., Natick, MA), NBNI (Research Biochemicals) and NTII (Research Biochemicals) were dissolved in 0.9% normal saline. DALCE (synthesized by Dr. W. D. Bowen NIDDK/NIH, Bethesda, MD) was dissolved in 0.2 M HCl in distilled water with the pH adjusted to 7.5 to 8.0 with 0.2 M NaOH. All antagonists were administered in 5 µl volumes over 30 sec. To allow for full development of irreversible antagonist effects equimolar (40 nmol) doses of FNA, NTII and DALCE were administered 24 hr before M6G administration, and an equimolar (40 nmol) dose of NBNI was administered 30 min before M6G administration (see review by Bodnar, 1996). All i.c.v. microinjections were infused at their prescribed volumes through a stainless steel internal cannula (28-gauge, Plastics One) connected to a Hamilton microsyringe by polyethylene tubing.

Protocol 1. All protocols were approved by the Queens College Institutional Animal Care and Use Committee. All rats in this and subsequent protocols were tested over a 4- to 10-day adaptation period at 3 to 9 hr into the light cycle to insure stability of baseline spontaneous food intake during this phase of the light cycle. Preweighed pellets were placed on the floor of the wire mesh cages to optimize accessibility because this factor can interfere with opioid-induced feeding (see review by Gosnell and Levine, 1996). Cumulative intakes were assessed 1, 2 and 4 hr after each condition, and were adjusted for spillage collected beneath each cage. After intake stabilization, five rats received a vehicle condition (Veh, 5 µl, 0.9% normal saline, i.c.v.). Because opioid agonists produce sedative and hypoactive effects (see review by Gosnell and Levine, 1996), each animal received two microinjections of M6G (500 ng, i.c.v.) without measuring intake. Then M6G doses of 10, 100, 500 and 1000 ng were administered to the five rats at weekly intervals, and food intake was assessed at 1, 2 and 4 hr later. That dose of M6G that increased food intake to a similar degree as the mu agonist, DAMGO (1 µg, i.c.v.) was chosen to allow comparison of antagonist and AS ODN effects relative to DAMGO-induced hyperphagia (Leventhal et al., 1997).

Protocol 2. After stabilization of intake and adaptation to potential sedative and hypoactive effects of M6G, subgroups of rats were exposed to the following conditions at weekly intervals: control (5 µl, 0.9% normal saline, i.c.v., n = 18), M6G (500 ng, n = 18), FNA at doses of 0.4 (n = 6), 4.0 (n = 7) and 40 (n = 7) nmol paired with M6G, NBNI (40 nmol) paired with M6G (n = 5), DALCE (40 nmol) paired with M6G (n = 5), and NTII (40 nmol) paired with M6G (n = 6). Cumulative food intake was assessed 4 hr after vehicle or M6G administration because this interval significantly and reliably increased M6G-induced intake (see "Results"). Rats receiving specific antagonists were matched for intake elicited by M6G alone. An additional group of six rats was evaluated to determine whether FNA (40 nmol) pretreatment altered intake (2 and 4 hr) after vehicle treatment.

Protocol 3. All rats were stabilized for intake and adaptation to potential sedative and hypoactive effects of each of the following opioid agonists. In assessing MOR-1 AS ODN effects upon M6G-induced hyperphagia, subgroups of rats received: 1) vehicle (5 µl, 0.9% normal saline, i.c.v., n = 31), 2) M6G (500 ng, i.c.v., n = 31), AS ODNs (10 µg, 2 µl, i.c.v.) directed against exons 3) 1 (AS1, n = 6), 4) 2 (AS2, n = 7), 5) 3 (AS3, n = 6) or 6) 4 (AS4, n = 6) of the MOR-1 clone before M6G and 7) a MS ODN directed against exon 2 of the MOR-1 clone (MS2, n = 6) before M6G. In assessing MOR-1 AS ODN effects upon morphine-induced hyperphagia, subgroups of rats received: 1) vehicle (n = 16), 2) morphine (5 µg, i.c.v., n = 16) and AS ODNs directed against exons 3) 1 (AS1, n = 8) or 4) 2 (AS2, n = 8) of the MOR-1 clone before morphine. In assessing DOR-1 AS ODN effects upon hyperphagia induced by either M6G or Delt II, subgroups of rats received: 1) vehicle (n = 10), 2) M6G (500 ng, n = 6), 3) Delt II (20 µg, n = 4) and an AS ODN directed against exon 3 of the DOR-1 clone before either 4) M6G (n = 6) or 5) Delt II (n = 4). In assessing KOR-1 AS ODN effects upon hyperphagia induced by either M6G or U50488H, subgroups of rats received: 1) vehicle (n = 10), 2) M6G (500 ng, n = 5), 3) U50488H (20 µg, n = 5), and an AS ODN directed against exon 3 of the KOR-1 clone prior to either 4) M6G (n = 5) or 5) U50488H (n = 5). In assessing KOR-3/ORL1 AS ODN effects upon hyperphagia induced by either M6G or nociceptin, subgroups of rats received: 1) vehicle (n = 13), 2) M6G (500 ng, n = 6), 3) nociceptin (18 µg, n = 7) and an AS ODN directed against exon 3 of the KOR-3/ORL1 clone before either 4) M6G (n = 6) or 5) nociceptin (n = 7). The 10 µg AS ODN dose was chosen because it was most effective in reducing M6G-induced analgesia (Rossi et al., 1997a). During the test phase of each protocol, rats received their particular AS ODN or MS ODN microinjection on days 1, 3 and 5 as previously described (Leventhal et al., 1996); this time course of treatment both down-regulates the synthesis of new receptors and permits turnover of existing receptors (see review by Pasternak and Standifer, 1995). Twenty-four hours after the last AS ODN treatment (day 6), rats were microinjected with either M6G, morphine, Delt II or U50488H, and food intake was assessed after 4 hr. The chosen dose and intake interval for each of these agonists were based on previous studies (e.g., Gosnell and Levine, 1996; Sanger and McCarthy, 1980; Yu et al., 1997). Intake was only measured after 2 hr after nociceptin given its shorter duration of action (Leventhal et al., 1998).

Statistics. Separate one-way analyses of variance were performed on cumulative food intake data at 1, 2 and 4 hr for the different doses of M6G (protocol 1), at 4 hr for the different antagonist treatments before M6G (protocol 2), and at 4 hr for the different AS ODN and MS ODN treatments before M6G and other agonists (protocol 3). Significant differences in intake measures were determined for each subgroup relative to both corresponding vehicle (Veh) control values and agonist conditions before antagonist or AS ODN treatments (Tukey comparisons, P < .05).

	Results

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M6G-induced hyperphagia. Significant differences in intake were observed across M6G doses after 2 [F(4,20) = 2.92, P < .047] and 4 (F = 29.02, P < .0001) hr, but not after 1 hr (F = 2.06, N.S.). M6G significantly and dose-dependently stimulated food intake after doses of either 100 ng after 4 hr, 500 ng after 2 and 4 hr and 1000 ng after 2 and 4 hr (fig. 1). In contrast, the 10-ng dose of M6G failed to alter intake at any time interval. Because the 500-ng dose of M6G produced the most comparable increase in intake relative to those doses of DAMGO used in a priori AS ODN study (Leventhal et al., 1997), this dose was used in antagonist and AS ODN paradigms.

View larger version (23K): [in this window] [in a new window]   Fig. 1.   Alterations (g, ± S.E.M.) in cumulative food intake after i.c.v. administration of the selective morphine metabolite, morphine 6-glucuronide (M6G). The asterisks in this and the subsequent figures indicate significant increases in M6G-induced feeding relative to corresponding vehicle control values (Tukey comparisons, P < .05).

Opioid antagonists and M6G-induced hyperphagia. Significant differences in intake were observed among equimolar antagonist conditions relative to vehicle and M6G treatment alone [F(5,53) = 15.58, P < .0001]. The significant increase in food intake after M6G after 4 hr was eliminated by pretreatment with a 40-nmol dose of the mu antagonist, FNA (fig. 2A). In contrast, equimolar doses of either delta₁, delta₂ or kappa₁ opioid antagonists failed to alter M6G-induced hyperphagia. In assessing the dose-dependent effects of FNA upon M6G-induced hyperphagia, significant differences were observed among conditions [F(4,37) = 7.92, P < .0001]. M6G-induced hyperphagia was significantly reduced by the 40, but not either the 0.4 or 4 nmol doses of FNA (fig. 2B). The 40-nmol dose of FNA failed to significantly alter intake following vehicle treatment [t(10) = 1.51, n.s.; data not shown].

View larger version (36K): [in this window] [in a new window]   Fig. 2.   A, Alterations (g, ± S.E.M.) in M6G- (500 ng, i.c.v.) induced hyperphagia (4 hr) following central pretreatment with an equimolar dose (40 nmol, i.c.v.) of either mu (FNA), delta1 (DALCE), delta2 (NTII) or kappa1 (NBNI) opioid antagonists. B, Alterations (g, ± S.E.M.) in M6G-induced hyperphagia (4 hr) in rats after central pretreatment with a dose-range of FNA (0.4-40 nmol, i.c.v.). The phi symbol in this and subsequent figures indicate significant decreases as compared to Veh-agonist values (Tukey comparisons, P < .05).

MOR-1 AS ODN treatment and M6G-induced hyperphagia. Significant differences in intake were observed among AS ODN and MS ODN conditions relative to vehicle and M6G treatment alone [F(6,84) = 15.18, P < .0001]. M6G-induced hyperphagia was differentially affected by MOR-1 AS ODN pretreatment such that it was significantly reduced by AS ODNs directed against either exons 2 (AS2: 66%) or 3 (AS3: 68%) of the MOR-1 clone (fig. 3A). In contrast, AS ODNs directed against either exons 1 (AS1) or 4 (AS4) of the MOR-1 clone failed to significantly affect M6G-induced hyperphagia. Further, the MS ODN control in which three bases had been changed in the effective exon 2 AS ODN sequence (table 1), failed to significantly alter M6G-induced hyperphagia (fig. 3A).

View larger version (36K): [in this window] [in a new window]   Fig. 3.   A, Alterations (g, ± S.E.M.) in M6G-induced hyperphagia (4 hr) after central pretreatment with AS ODNs directed against either exons 1 (AS1), 2 (AS2), 3 (AS3) or 4 (AS4) of the MOR-1 clone or a missense ODN (MS2). B, Alterations (g, ± S.E.M.) in morphine- (5 µg, i.c.v.) induced hyperphagia (4 hr) after central pretreatment with AS ODNs directed against either exons 1 (AS1) or 2 (AS2) of the MOR-1 clone.

MOR-1 AS ODN treatment and morphine-induced hyperphagia. Significant differences in intake were observed among AS ODN conditions relative to vehicle and morphine treatment alone [F(3,27) = 14.79, P < .0001]. Morphine-induced hyperphagia was differentially affected by MOR-1 AS ODN pretreatment such that it was significantly reduced by the AS ODN directed against exon 1 (AS1: 59%), but not exon 2 (AS2) of the MOR-1 clone (fig. 3B). This pattern of effects coincides with MOR-1 AS ODN effects upon DAMGO-induced hyperphagia (Leventhal et al., 1997), and is distinct from that pattern observed for M6G-induced hyperphagia.

DOR-1 AS ODN treatment, M6G and Delt II. In assessing DOR-1 AS ODN effects upon M6G-induced hyperphagia, significant differences in intake were observed among conditions [F(2,19) = 9.75, P < .0012]. An AS ODN directed against exon 3 of the DOR-1 clone failed to significantly alter M6G-induced hyperphagia (fig. 4A). To establish the activity of this probe, its effects upon Delt II-induced hyperphagia were evaluated. The significant difference in intake among conditions [F(2,15) = 15.47, P < .0002] revealed that Delt II-induced hyperphagia was significantly reduced by 77% following the AS ODN probe directed against exon 3 of the DOR-1 clone (fig. 4A).

View larger version (25K): [in this window] [in a new window]   Fig. 4.   A, Alterations (g, ± S.E.M.) in hyperphagia (4 hr) induced by M6G (500 ng, i.c.v.) or the selective delta2 opioid agonist, Delt II (20 µg, i.c.v.) after central pretreatment with an AS ODN probe directed against exon 3 of the DOR-1 clone. B, Alterations (g, ± S.E.M.) in hyperphagia (4 hr) induced by M6G or the selective kappa1 opioid agonist, U50,488H (20 µg, i.c.v.) after central pretreatment with an AS ODN probe directed against exon 3 of the KOR-1 clone. C, Alterations (g, ± S.E.M.) in hyperphagia induced by M6G (4 hr) or the selective endogenous peptide for the KOR-3/ORL-1 clone, nociceptin (18 µg, i.c.v., 2 hr) after central pretreatment with an AS ODN probe directed against exon 3 of the KOR-3/ORL1 clone.

KOR-1 AS ODN treatment, M6G and U50488H. In assessing KOR-1 AS ODN effects upon M6G-induced hyperphagia, significant differences in intake were observed among conditions [F(2,17) = 9.29, P < .0019]. An AS ODN directed against exon 3 of the KOR-1 clone failed to significantly alter M6G-induced hyperphagia (fig. 4B). To establish the activity of this probe, its effects on U50488H-induced hyperphagia were evaluated. The significant difference in intake among conditions [F(2,17) = 12.31, P < .0005] revealed that U50488H-induced hyperphagia was significantly reduced by 82% after the AS ODN probe directed against exon 3 of the KOR-1 clone (fig. 4B).

KOR-3/ORL1 AS ODN treatment, M6G and nociceptin. In assessing KOR-3/ORL1 AS ODN effects on M6G-induced hyperphagia, significant differences in intake were observed among conditions [F(2,22) = 30.69, P < .0001]. An AS ODN directed against exon 3 of the KOR-3/ORL1 clone failed to significantly alter M6G-induced hyperphagia (fig. 4C). To establish the activity of this probe, its effects on nociceptin-induced hyperphagia were evaluated. The significant difference in intake among conditions [F(2,24) = 12.96, P < .0002] revealed that nociceptin-induced hyperphagia was eliminated following the AS ODN probe directed against exon 3 of the KOR-3/ORL1 clone (fig. 4C).

	Discussion

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Our findings confirmed the major goals of the study. First, centrally administered M6G significantly and dose-dependently increased spontaneous food intake, and is the first reported observation of a hyperphagic response elicited by this morphine metabolite. The increased intake was gradual, occurring 2 and 4 hr after M6G administration, but failing to increase intake after 1 hr. This temporal pattern of ingestive effects is commonly observed after administration of such other opiates as heroin, butorphanol, codeine and levorphanol (e.g., Levine and Morley, 1983; Levine et al., 1994; Sanger and McCarthy, 1980; Thornhill et al., 1976). M6G also produced clear dose-dependent actions with low (10 ng) doses failing to increase intake, and higher doses (100-1000 ng) systematically increasing intake. The effective dose range of M6G to induce feeding is more than 10-fold lower than comparable morphine doses after ventricular administration (see review by Gosnell and Levine, 1996). Thus, the relationship between the respective potencies of M6G-induced hyperphagia relative to morphine-induced hyperphagia is similar to the relationship between their respective potencies to elicit analgesic responses (Abbott and Palmour, 1988; Frances et al., 1992; Pasternak et al., 1987; Paul et al., 1989; Shimomura et al., 1971; Sullivan et al., 1989). These behavioral and functional differences appear to persist even though M6G labels mu receptors with an affinity slightly less than morphine in binding assays (Paul et al., 1989).

Selective opioid antagonists differentially altered the magnitude of M6G-induced hyperphagia. The selective, irreversible mu opioid receptor antagonist, FNA significantly and dose-dependently decreased M6G-induced hyperphagia, and almost blocked its expression after the highest antagonist dose. FNA exerted this inhibitory action upon M6G-induced hyperphagia without altering the low levels of spontaneous intake after vehicle treatment. M6G-induced hyperphagia was unaffected by pretreatment with an equimolar dose of the kappa₁ opioid receptor antagonist, NBNI that distinguishes this ingestive response from other forms of mu-mediated hyperphagia (Levine et al., 1990, 1991). Further, an equimolar dose of either delta₁ (DALCE) or delta₂ (NTII) opioid receptor antagonists failed to alter the magnitude of M6G-induced hyperphagia. These data strongly suggest that M6G-induced hyperphagia is acting through selective activation of pharmacologically characterized mu opioid receptors. However, it is important to determine whether equimolar doses of each antagonist produce equal degrees of blockade of functional responses at the intended receptor subtype targets. FNA at doses of 0.1 to 10 nmol significantly decreased DAMGO-induced feeding by 57 to 71%, but failed to alter intake induced by kappa (U50488H) or delta (D-Ser², Leu⁵, Thr⁶-enkephalin: DSLET) agonists (Levine et al., 1991). Our data indicated that FNA at 40 nmol, but not at 0.4 or 4 nmol, significantly reduced M6G-induced hyperphagia. A comparable dose of FNA (50 nmol) also decreased butorphanol-induced feeding (Levine et al., 1994). It is conceivable that this higher FNA dose could alter kappa- and delta-mediated responses. NBNI at doses of 1, 10 and 100 nmol significantly decreased feeding induced by kappa (U50488H, butorphanol), delta (DSLET) and mu (DAMGO) agonists (Levine et al., 1990, 1994). Therefore, the failure of NBNI at a 40-nmol dose to alter M6G-induced feeding suggests that the ineffectiveness of the antagonist indicates a lack of involvement of the kappa receptor in M6G-induced feeding. Similarly, the inability of the 40 nmol dose of delta₁ (DALCE) or delta₂ (NTII) opioid antagonists to alter M6G-induced hyperphagia suggests a lack of involvement of these receptors in M6G-induced feeding since comparable doses of these antagonists respectively block intake elicited by delta₁ (D-Pen², D-Pen⁵-enkephalin) and delta₂ (deltorphin II) agonists (Yu et al., 1997). The above data underscore the limitations in interpreting antagonist data, namely the need to use equimolar doses of antagonists paired with the ability of such doses to exert functional effects at their respective receptors. Although our study generally satisfied both of these criteria, further converging evidence was needed to assess the receptor mediation of M6G-induced feeding. The use of antisense probes provided such support for mu receptor mediation of this response.

The differences in the mediation of M6G-induced hyperphagia relative to DAMGO-induced and morphine-induced hyperphagia were characterized further by our AS ODN studies (table 2). We (Leventhal et al., 1996) previously found that spontaneous intake and body weight were significantly reduced by AS ODNs directed against each of the four exons of the MOR-1 clone. Rossi and coworkers (1995a, 1995b, 1997a) demonstrated that the actions of AS ODNs directed against exons 2 and 3 of the MOR-1 clone that blocked M6G-induced analgesia were distinct from the actions of AS ODNs directed against exons 1 and 4 of the MOR-1 clone, which blocked analgesia elicited by morphine and DAMGO. We (Leventhal et al., 1997) recently found that DAMGO-induced hyperphagia displays an identical pattern of sensitivity to AS ODNs directed against the MOR-1 clone to that observed for morphine and DAMGO-induced analgesia. The present study found that M6G-induced hyperphagia was reduced by AS ODNs directed against either exon 2 or exon 3 of the MOR-1 clone, although AS ODNs directed against either exons 1 or 4 of this clone were ineffective. The pattern of MOR-1 AS ODN effects on M6G-induced hyperphagia was specific to the AS ODN sequence because an MS ODN which differed from an effective AS ODN probe by changing three nucleotide bases, failed to alter M6G-induced hyperphagia. Our study further demonstrated that the pattern of MOR-1 AS ODN effects on morphine-induced hyperphagia was similar to that observed for DAMGO-induced hyperphagia, and distinct from the hyperphagic responses of its active metabolite, M6G. Thus, an AS ODN directed against exon 1 of the MOR-1 clone significantly reduced morphine-induced hyperphagia, whereas the probe directed against exon 2 of the MOR-1 clone failed to exert significant effects. One potential limitation in interpreting these data was the use of a single dose (10 µg) of each AS ODN probe. This fixed dose was chosen because higher (25 µg) AS ODN doses have been shown to produce nonspecific reductions in morphine-induced analgesia such that these higher dose-dependent AS ODN effects persist well after the termination (7 days) of AS ODN treatment (Rossi et al., 1997a). In contrast, the chosen (10 µg) dose produces potent reductions in morphine-induced analgesia that recover in a manner consistent with receptor turnover and synthesis.

Despite these important dissociations and the positive controls used in in vivo testing, it is important to determine whether there are changes in the transcriptional and translational products of the genes in question. However, there are crucial limitations in discerning whether the binding of DAMGO or morphine relative to M6G in vitro are altered by the different AS ODN treatments. The levels of high affinity M6G binding in the brain is only 10% of total mu opioid receptor binding (Brown et al., 1997b). Therefore, even in the event that a particular MOR-1 AS ODN treatment (e.g., exon 2 or exon 3) completely eliminated M6G binding, detection of such changes would be difficult as a function of total mu opioid receptor binding. Further, AS ODN administration generally only produces modest (40%) reductions in receptor protein levels for opioid receptors (Pasternak and Standifer, 1995), thus making the changes in M6G binding by AS ODN treatment even more difficult to detect. Another possibility for such differences may be due to changes in signaling for G-protein coupling for one ligand relative to the other. Although there is differential blockade of opioid analgesia by AS ODNs directed against various G-protein subunits (Standifer et al., 1996), it is not known whether these substrates mediate the observed effects. These provisos need to be considered despite our in vivo positive controls. However, these data appear to provide converging evidence for mu (MOR-1) mediation of M6G-induced hyperphagia, and the exons subserving this response are both distinct from traditional mu agonists in both analgesic and hyperphagic assays yet identical to those exons subserving M6G-induced analgesia.

The involvement of opioid receptor clones in mediating M6G-induced hyperphagia was limited to the MOR-1 clone because AS ODN probes directed against either the DOR-1, KOR-1 or KOR-3/ORL1 clones failed to alter M6G-induced hyperphagia. Our study demonstrated conclusively that the failure of these probes was due to their lack of inherent involvement in M6G-induced hyperphagia, and not because these probes lacked intrinsic activity in hyperphagic assays. Thus, an AS ODN directed against exon 3 of the DOR-1 clone significantly reduced hyperphagia induced by the selective delta₂ opioid agonist, Delt II. Such inhibition is of interest because it has been suggested that the DOR-1 clone gene encodes the pharmacologically characterized delta₂ opioid receptor subtype. This DOR-1 AS ODN effect parallels actions observed in analgesic assays for Delt II (Rossi et al., 1997b). Further, an AS ODN directed against exon 3 of the KOR-1 clone significantly reduced hyperphagia induced by the selective kappa₁ opioid agonist, U50488H. This KOR-1 AS ODN effect parallels actions observed in analgesic assays for U50488H (Chien et al., 1994). Finally, an AS ODN directed against exon 3 of the KOR-3/ORL1 clone significantly reduced hyperphagia induced by the non-traditional opioid peptide, nociceptin/orphanin FQ (Meunier et al., 1995; Reinscheid et al., 1995). Nociceptin has little affinity for traditional opioid receptors and its actions through the KOR-3/ORL1 clone has been confirmed in both hyperphagic and analgesic assays (Leventhal et al., 1998; Rossi et al., 1998). It should be noted that whereas effective doses of each of these agonists elicit significant hyperphagia in each animal, the magnitude of some of these agonists were not equivalent to that induced by M6G. Clear dose-response relationships are often problematic for opioid-induced hyperphagia because they produce sharp step-wise functions (Gosnell and Levine, 1996). Yet these data provide novel evidence indicating that hyperphagic responses induced by Delt II, U50488H and nociceptin are mediated respectively by the DOR-1, KOR-1 and KOR-3/ORL1 clones. These data further indicate the selectivity of mu and MOR-1 opioid actions in the mediation of M6G-induced hyperphagia.

It is now clear that a single receptor is not responsible for the common drug actions of morphine and M6G across a number of functional assays. Specifically, the similarity in the AS ODN profile of the hyperphagic and analgesic responses to M6G, relative to the AS ODN profile of the hyperphagic and analgesic responses to traditional mu receptor agonists lend credence to the concept of a novel M6G receptor. The persistence of M6G-induced analgesia in mu-deficient CXBK mice, morphine-tolerant mice and transgenic mice with disruption of exon 1 of the MOR-1 gene provides further support for the existence of a novel M6G receptor (Brown et al., 1997a, 1997b; Rossi et al., 1996; Schuller et al., 1997). Such a receptor could conceivably result from alternative splice variants of the MOR-1 clone, although a distinct gene cannot be ruled out.