Multiple opioid receptors mediate feeding elicited by mu and delta opioid receptor subtype agonists in the nucleus accumbens shell in rats

Brain Research 876 (2000) 76–87 www.elsevier.com / locate / bres

Research report

Multiple opioid receptors mediate feeding elicited by mu and delta opioid receptor subtype agonists in the nucleus accumbens shell in rats Andre´ Ragnauth, Malgorzata Moroz, Richard J. Bodnar* Department of Psychology and Neuropsychology Doctoral Sub-Program, Queens College, City University of New York, 65 -30 Kissena Blvd., Flushing, NY 11367, USA Accepted 20 June 2000

Abstract The nucleus accumbens, and particularly its shell region, is a critical site at which feeding responses can be elicited following direct administration of opiate drugs as well as m-selective and d-selective, but not k-selective opioid receptor subtype agonists. In contrast to observations of selective and receptor-specific opioid antagonist effects upon corresponding agonist-induced actions in analgesic studies, ventricular administration of opioid receptor subtype antagonists blocks feeding induced by multiple opioid receptor subtype agonists. The present study examined whether feeding responses elicited by either putative m ([D-Ala 2 , NMe-Phe 4 , Gly-ol 5 ]-enkephalin (DAMGO)), d 1 ([D-Pen 2 , D-Pen 5 ]-enkephalin (DPDPE)) or d 2 ([D-Ala 2 , Glu 4 ]-deltorphin (Deltorphin)) opioid receptor subtype agonists administered into the nucleus accumbens shell were altered by accumbens pretreatment with either selective m (b-funaltrexamine), m 1 (naloxonazine), d 1 ([D-Ala 2 , Leu 5 , Cys 6 ]-enkephalin (DALCE)), d 2 (naltrindole isothiocyanate) or k 1 (nor-binaltorphamine) opioid receptor subtype antagonists. Similar magnitudes and durations of feeding responses were elicited by bilateral accumbens administration of either DAMGO (2.5 mg), DPDPE (5 mg) or Deltorphin (5 mg). DAMGO-induced feeding in the nucleus accumbens shell was significantly reduced by accumbens pretreatment of m, d 1 , d 2 and k 1 , but not m 1 opioid receptor subtype antagonists. DPDPE-induced feeding in the accumbens was significantly reduced by accumbens pretreatment of m, d 1 , d 2 and k 1 , but not m 1 opioid receptor subtype antagonists. Deltorphininduced feeding in the accumbens was largely unaffected by accumbens d 2 antagonist pretreatment, and was significantly enhanced by accumbens m or k 1 antagonist pretreatment. These data indicate different opioid pharmacological profiles for feeding induced by putative m, d 1 and d 2 opioid agonists in the nucleus accumbens shell, as well as the participation of multiple opioid receptor subtypes in the elicitation and maintenance of feeding by these agonists in the nucleus accumbens shell.  2000 Elsevier Science B.V. All rights reserved. Theme: Neurotransmitters, modulators, transporters and receptors Topic: Opioids: anatomy, physiology and behavior Keywords: Feeding; Nucleus accumbens shell; m Opioid receptor; m 1 Opioid receptor; d 1 Opioid receptor; d 2 Opioid receptor; k 1 Opioid receptor

1. Introduction The nucleus accumbens (NAcc) is one of a number of structures that supports increases in spontaneous food intake following microinfusions of opioid agonists (see review in Ref. [11]). Thus, the prototypical opioid agonist, morphine stimulates food intake following administration *Corresponding author. Tel.: 11-718-997-3543; fax: 11-718-9973257. E-mail address: richard [email protected] (R.J. Bodnar). ]

into the NAcc [2,3,10,30,37]. Receptor autoradiographic and mRNA gene expression studies have verified that m, d and k opioid receptors can be found in the NAcc [14,28,32,33]. In assessing the sensitivity of selective opioid receptor subtype agonists in the NAcc, Bakshi and Kelley [1] observed that spontaneous feeding was elicited following administration of the selective m opioid agonist, DAMGO, or the selective d 1 opioid agonist, DPDPE, but not the selective k 1 opioid agonist, U50488H in the shell region of the NAcc. Similarly, DAMGO and DPDPE, but not U50488H or

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dynorphin, enhanced sucrose intake in the NAcc [71]. Moreover, DAMGO microinjected into the NAcc selectively increased intake of a fat relative to a carbohydrate diet [70]. Further support for the presence of m-mediated ingestive actions within the NAcc included the observations that the selective m opioid antagonist, b-funaltrexamine (b-FNA), significantly reduced feeding responses under either food deprivation, 2-deoxy-D-glucose glucoprivation or palatable sucrose conditions [5,21]. In contrast, the m 1 opioid antagonist, naloxonazine (NAZ), failed to exert effects, suggesting that the low-affinity m 2 opioid binding site (see review in Ref. [41]) was responsible for these effects within the NAcc. Actions of the k 1 opioid antagonist, nor-binaltorphamine (NBNI) in the NAcc were largely limited to potent reductions in glucoprivic intake [5], while d receptor antagonism with naltrindole in the NAcc actually potentiated intake under deprivation and palatable conditions [21]. While DAMGO has been generally accepted as a mselective opioid agonist (e.g. Ref. [54]), pharmacological and biochemical data have suggested the existence of d opioid receptor subtypes termed d 1 and d 2 [17– 19,34,35,38,55]. These distinctions were confirmed in analgesic assays such that the enkephalin analogue, DPDPE [36] acts as a d 1 -opioid receptor agonist based upon its sensitivity to selective d 1 opioid antagonism by DALCE, but not to selective d 2 opioid antagonism by naltrindole isothiocyanate (NTII) [19,55]. In contrast, analgesia elicited by the d 2 opioid agonist, Deltorphin [23], is sensitive to d 2 , but not d 1 opioid antagonism [19,34,47,55]. Further, analgesic cross-tolerance fails to occur between DPDPE and Deltorphin [35]. Moreover, supraspinal analgesia elicited from the ventrolateral periaqueductal gray and the rostral ventromedial medulla is observed following Deltorphin, but not DPDPE [6,51]. Indeed, distinctions between analgesic responses elicited by DAMGO and Deltorphin can be made in these sites such that the former response is blocked by m, but not d opioid antagonism, whereas the latter response is blocked by d, but not m opioid antagonism [51]. These data suggest receptor selectivity and specificity in producing analgesic responses elicited by opioid receptor subtype agonists. Feeding responses induced by different opioid receptor subtype agonists do not produce this clean-cut specificity between opioid receptor subtype agonists and antagonists. Thus, ventricular administration of the k 1 antagonist, NBNI significantly reduced feeding induced by the k 1 agonist, U50488H, but not by the k 3 agonist, naloxone benzoylhydrazone [22,27]. However, Levine and co-workers subsequently demonstrated that effective doses of NBNI also decreased feeding elicited by either DAMGO or the preferential d agonist, [D-Ser 2 , Leu 5 , Thr 6 ]-enkephalin (DSLET). Although NBNI was initially characterized as a selective k antagonist [45], further studies using chronic NBNI injections indicated m and d activity as well [56]. Whereas b-FNA potently reduces feeding induced by

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ventricular administration of the m agonist, DAMGO [25,26], this antagonist also reduces feeding induced by ventricular administration of the d agonist, DSLET, but not the k 1 agonist, U50488H. Similar separations between selective agonist and antagonist effects occurred for feeding responses induced by d 1 and d 2 mechanisms. Thus, feeding induced by ventricular administration of the d 1 agonist, DPDPE, was blocked by general opioid antagonism with naltrexone and d 2 antagonism with NTII, but not by d 1 antagonism with DALCE [69]. Alternatively, feeding induced by ventricular administration of the d 2 agonist, Deltorphin was blocked by d 1 (DALCE) and d 2 (NTII) antagonist pretreatment, but not by the general opioid antagonist, naltrexone [69]. Given that ventricular routes of administration were used in all of the above studies, it is possible that some of the multiple and anomalous results may be due to actions at multiple opioid receptor sites. Therefore, the present study had the following aims. Firstly, our laboratory wanted to determine whether Deltorphin would produce feeding following microinjection into the shell region of the NAcc at similar dose levels and with similar magnitudes to that observed for DAMGO and DPDPE [1,70,71]. Secondly, our laboratory examined whether feeding induced by either DAMGO, DPDPE or Deltorphin in the shell region of the NAcc were mediated through single or multiple opioid receptor subtypes by pretreating rats in the shell region of the NAcc with selective m (b-FNA), m 1 (NAZ), d 1 (DALCE), d 2 (NTII) or k 1 (NBNI) opioid receptor antagonists. The shell region of the NAcc was chosen for study because of the greater effectiveness of m and d agonists to elicit feeding from this area relative to either the core region of the NAcc or the surrounding ventral striatum [1,2]. A preliminary version of these findings has been presented [48].

2. Materials and methods

2.1. Subjects, surgery and histology Adult male albino Sprague–Dawley rats (Charles River Laboratories, Kingston, NY, 80–120 days of age) were individually housed in wire-mesh cages and maintained on a 12 h light / 12 h dark cycle with Purina standard laboratory rat chow and water available ad libitum. Each rat was pretreated with chlorpromazine (3 mg / kg, i.p.) and anesthetized with ketamine HCl (120 mg / kg, i.p.). Bilateral stainless steel guide cannulae (26 gauge, Plastics One, Roanoke, VA) were aimed stereotaxically (Kopf Instruments, Tujunda, CA) at the shell region of the NAcc using the following coordinates: incisor bar (15), 3.5 mm anterior to the bregma suture, 1.7 mm lateral to and angled 108 toward either side of the sagittal suture and 6.4–6.6 mm from the top of the skull. The cannulae were secured to the skull by four anchor screws with dental acrylic. To allow full drug clearance, all animals were allowed at least

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2 weeks to recover from stereotaxic surgery before behavioral testing began. At the completion of testing, all animals were injected into the heart with potassium chloride (15 mg / 1 ml 0.9% saline), and following anesthetization, were perfused transcardially with 0.9% normal saline followed by 10% buffered formalin. Coronal 40-mm sections, stained with Cresyl violet, were examined by light microscopy by an observer unfamiliar with the behavioral data; only animals with confirmed cannulae placements were included in the data analysis.

2.2. Drugs The opioid agonists, DAMGO (2.5 mg), DPDPE (5 mg) and Deltorphin (5 mg), were obtained from Peninsula Laboratories (Belmont, CA) and were dissolved in a 0.9% normal saline solution. The opioid antagonists, b-FNA and NBNI, were obtained from Research Biochemicals International (Natick, MA) and were dissolved in a 0.9% normal saline solution. DALCE (synthesized by Dr. W.D. Bowen) was dissolved in 0.2 M HCl in distilled water with the pH raised to 7.5 by adding 0.2 M NaOH. NAZ (synthesized by Dr. G.W. Pasternak) was dissolved in distilled water and 0.2% glacial acetic acid. Bilateral microinjections delivered half of the total dose of agonists and antagonists to the left and right shell region of the NAcc. Antagonist treatments, reflecting peak dose and time intervals, preceded agonist treatments by 1 h for NBNI, and by 24 h for b-FNA, NAZ, DALCE and NTII. On the test day at 3–5 h into the light cycle, food was removed from the cages and antagonist-treated animals received the particular opioid agonist. All microinjections were administered bilaterally in 1-ml volumes over 30 s through a stainless steel internal cannula (33-gauge, Plastics One) connected to a Hamilton microsyringe by polyethylene tubing. This relatively high injection volume was necessary because of limited solubility of some of the antagonists.

2.3. Protocols In all experiments, each of 56 rats was initially tested for intake over a 4-h time course following vehicle treatment. Then each rat was tested for intake over a 4-h time course following administration of either DAMGO (n522), DPDPE (n516) or Deltorphin (n518). Only rats that displayed at least two consecutive sessions of agonistinduced feeding were included in the antagonist paradigm; these animals did not display either increments (sensitization) or decrements (adaptation) in their agonist-induced ingestive responses. Subgroups of rats receiving different antagonist conditions for each agonist paradigm were matched for intake elicited by that particular agonist. These subgroups of rats received between one and four additional paired antagonist–agonist injection conditions according to a counterbalanced design. In assessing opioid receptor subtype antagonism upon m-opioid agonist-in-

duced feeding, 22 rats received the following bilateral microinjection conditions: (a) vehicle (n522), (b) DAMGO (n522), b-FNA at doses of either (c) 0.1 (n57), (d) 1 (n510) or (e) 4 (n59) mg paired with DAMGO, (f) NAZ at a 4-mg dose paired with DAMGO (n59), DALCE at doses of either (g) 1 (n57) or (h) 4 (n510) mg paired with DAMGO, NTII at doses of either (i) 1 (n57) or (j) 4 (n58) mg paired with DAMGO, and NBNI at doses of either (k) 1 (n512) or (l) 4 (n58) mg paired with DAMGO. In assessing opioid receptor subtype antagonism upon d 1 -opioid agonist-induced feeding, 16 rats received the following bilateral microinjection conditions: (a) vehicle (n516), (b) DPDPE (n516), b-FNA at doses of either (c) 1 (n58) or (d) 4 (n57) mg paired with DPDPE, (e) NAZ at a 4-mg dose paired with DPDPE (n57), DALCE at doses of either (f) 1 (n57) or (g) 4 (n58) mg paired with DPDPE, NTII at doses of either (h) 1 (n57) or (i) 4 (n57) mg paired with DPDPE, and NBNI at doses of either (j) 1 (n59) or (k) 4 (n57) mg paired with DPDPE. In assessing opioid receptor subtype antagonism upon d 2 -opioid agonist-induced feeding, 18 rats received the following bilateral microinjection conditions: (a) vehicle (n517), (b) Deltorphin (n517), b-FNA at doses of either (c) 1 (n57) or (d) 4 (n57) mg paired with Deltorphin, NTII at doses of either (e) 1 (n59), (f) 4 (n57) or (g) 8 (n58) mg paired with Deltorphin, and (h) NBNI at a 4-mg dose paired with Deltorphin (n58). It should be noted that neither NAZ nor DALCE was available for the last part of this protocol, and therefore these antagonists were not tested against Deltorphin-induced feeding. In all conditions, cumulative intakes were assessed at 1, 2 and 4 h after the last injection by measuring preweighed standard laboratory rat chow pellets which was adjusted for spillage collected by paper towels placed under the wire mesh cages.

2.4. Statistical analysis Separate one-way completely randomized analyses of variance were performed at each cumulative intake point for each feeding paradigm. Tukey corrected comparisons (P,0.05) were used to assess significant alterations in agonist-induced feeding relative to corresponding control conditions, and Fisher exact test comparisons (corrected t, P,0.05) evaluated significant alterations induced by antagonist pretreatment upon agonist-induced feeding relative to corresponding agonist-induced feeding per se.

3. Results

3.1. Histological verification Fig. 1 is a schematic of representative bilateral guide cannulae placements into the NAcc shell. Cannulae placements were all localized to the NAcc shell as far rostral as

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Fig. 1. Schematic depiction of directions of bilateral guide cannulae placements positioned towards the shell region of the nucleus accumbens (AcbSh). The majority of the tips of the confirmed cannula placements were found in the shaded area. All placements were found medial to the core region of the nucleus accumbens (AcbC) and lateral to the ventral diagonal band (VNB) area. aca, anterior commissure. This schematic was based upon the Paxinos and Watson Brain Atlas [42].

Figure 9, and as far caudal as Figure 14 of the Paxinos and Watson atlas [42]. The blackened area represents the multiple locations of cannula tips in the NAcc shell.

3.2. NAcc opioid receptor subtype antagonists and NAcc DAMGO-induced feeding Significant differences in spontaneous food intake were observed among injection conditions after 1 (F(11,119)5 5.11, P,0.0001), 2 (F55.87, P,0.0001) and 4 (F55.63, P,0.0001) h. DAMGO significantly increased food intake relative to control treatment after 1, 2 and 4 h (Figs. 2 and 3). Pretreatment in the NAcc with the selective m opioid receptor subtype antagonist, b-FNA, dose-dependently reduced DAMGO-induced feeding across the 4-h time course (Fig. 2A). Whereas rats pretreated with the 0.1-mg dose of b-FNA displayed normal DAMGO-induced feeding in the NAcc, rats pretreated with either 1- or 4-mg doses of b-FNA significantly reduced DAMGO-induced feeding in the NAcc over the 4-h time course. In contrast, pretreatment with the selective m 1 opioid receptor subtype antagonist, NAZ in the NAcc failed to alter NAcc DAMGO-induced feeding at any time point (Fig. 2B). Pretreatment with the higher, but not lower dose of the selective d 1 opioid receptor subtype antagonist, DALCE, in the NAcc significantly reduced NAcc DAMGO-induced feeding after 4 h (Fig. 3A). NAcc DAMGO-induced feeding was dosedependently reduced by pretreatment with the selective d 2 opioid receptor antagonist, NTII in the NAcc with the 4, but not the 1 mg antagonist dose, thereby preventing the expression of NAcc DAMGO-induced feeding after 2 and 4 h (Fig. 3B). Similarly, pretreatment of the selective k 1

opioid receptor subtype antagonist, NBNI, in the NAcc significantly reduced NAcc DAMGO-induced feeding following the 4 but not the 1-mg dose across the 4-h time course (Fig. 3C). Therefore, it appears that the m, d 1 , d 2 and k 1 opioid receptor antagonists, but not a m 1 opioid receptor antagonist administered into the NAcc block feeding induced by the m opioid agonist, DAMGO in the NAcc.

3.3. NAcc opioid receptor subtype antagonists and NAcc DPDPE-induced feeding Significant differences in spontaneous food intake were observed among injection conditions after 1 (F(10,88)5 3.90, P,0.0002), 2 (F53.38, P,0.0009) and 4 (F53.52, P,0.0006) h. DPDPE significantly increased food intake relative to control treatment after 1, 2 and 4 h (Figs. 4 and 5). Pretreatment in the NAcc with the selective m opioid receptor subtype antagonist, b-FNA, significantly and dose-dependently reduced DPDPE-induced feeding with the higher dose effective after 1 and 2 h (Fig. 4A). In contrast, pretreatment with the selective m 1 opioid receptor subtype antagonist, NAZ in the NAcc failed to alter NAcc DPDPE-induced feeding at any time point (Fig. 4B). Pretreatment with the selective d 1 opioid receptor subtype antagonist, DALCE, in the NAcc significantly and dosedependently reduced NAcc DPDPE-induced feeding with the higher dose effective after 4 h (Fig. 5A). Pretreatment with the selective d 2 opioid receptor antagonist, NTII in the NAcc unexpectedly and significantly reduced NAcc DPDPE-induced feeding after 4 h following the 1, but not the 4 mg antagonist dose (Fig. 5B).

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Fig. 2. Alterations in spontaneous food intake (g, 6S.E.M.) following a 2.5-mg dose of the m-selective opioid agonist, DAMGO (DAM) bilaterally in the NAcc shell relative to control treatment. The upper and lower panels depict effects upon DAMGO-induced feeding following pretreatment (24 h) with different doses (mg) of either the selective m-opioid antagonist, b-funaltrexamine (b-FNA) or the selective m 1 -opioid antagonist, naloxonazine (NAZ), respectively. The asterisks (*) in this and subsequent figures indicate significant increases in food intake following opioid agonist treatment relative to control treatment (Tukey comparisons, P,0.05). The crosses (1) in this and subsequent figures indicate significant alterations in food intake following opioid antagonist pretreatment relative to opioid agonist treatment alone (Tukey comparisons, P,0.05).

Pretreatment with the selective k 1 opioid receptor antagonist, NBNI in the NAcc significantly and dosedependently reduced DPDPE-induced feeding after 4 h following the 4, but not the 1 mg antagonist dose (Fig. 5C). Thus it appears that m, d 1 , d 2 and k 1 , but not m 1 opioid receptor antagonists injected into the NAcc block feeding induced by the d 1 opioid agonist, DPDPE in the NAcc.

3.4. NAcc opioid receptor subtype antagonists and Deltorphin-induced feeding Significant differences in spontaneous food intake were observed among injection conditions after 1 (F(7,74)5 6.12, P,0.0001), 2 (F56.58, P,0.0001) and 4 (F54.66, P,0.0002) h. Deltorphin significantly increased food

intake relative to control treatment after 1, 2 and 4 h (Fig. 6). Pretreatment with the selective d 2 opioid receptor subtype antagonist, NTII, in the NAcc failed to alter NAcc Deltorphin-induced feeding at any time point and at any on the antagonist doses ranging from 1 to 8 mg (Fig. 6A). Pretreatment with the selective m opioid receptor subtype antagonist, b-FNA, in the NAcc, failed to alter NAcc Deltorphin-induced feeding except for a transient (1 h) though significant increase in intake following the 4 mg antagonist dose (Fig. 6B). Indeed, pretreatment with the selective k 1 opioid receptor subtype antagonist, NBNI, in the NAcc, significantly increased NAcc Deltorphin-induced feeding after 1 and 2 h (Fig. 6C). Thus, it appears that none of the selective opioid receptor subtype antagonists tested decreased Deltorphin-induced feeding in the NAcc, and that m and k 1 opioid antagonist pretreatment augmented this ingestive response.

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Fig. 3. Alterations in spontaneous food intake (g, 6S.E.M.) following a 2.5-mg dose of the m-selective opioid agonist, DAMGO (DAM) bilaterally in the NAcc shell relative to control treatment. The upper and middle panels depict effects upon DAMGO-induced feeding following pretreatment (24 h) with different doses (mg) of either the selective d 1 -opioid antagonist, DALCE or the selective d 2 -opioid antagonist, naltrindole isothiocyanate (NTII), respectively. The lower panel depicts effects upon DAMGO-induced feeding following pretreatment (1 h) with different doses (mg) of the selective k 1 -opioid antagonist, nor-binaltorphamine (NBNI).

4. Discussion Similar magnitudes and time courses of feeding responses were elicited from the NAcc following microinjections of either the m-selective opioid agonist, DAMGO, the

d 1 -selective opioid agonist, DPDPE, or the d 2 -selective opioid agonist, Deltorphin. In contrast to intracerebral and supraspinal analgesic studies in which antagonist-specific effects produced selective blockade of analgesic responses following either DAMGO, DPDPE or Deltorphin [17–

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Fig. 4. Alterations in spontaneous food intake (g, 6S.E.M.) following a 5-mg dose of the d 1 -selective opioid agonist, DPDPE (DPD) bilaterally in the NAcc shell relative to control treatment. The upper and lower panels depict effects upon DPDPE-induced feeding following pretreatment (24 h) with different doses (mg) of either the selective m-opioid antagonist, b-funaltrexamine (b-FNA) or the selective m 1 -opioid antagonist, naloxonazine (NAZ), respectively.

19,34,35,51,55], selective antagonists for different opioid receptor subtypes exerted effects upon multiple opioid agonist-induced feeding responses within the NAcc. Firstly, the fact that DAMGO, like morphine, elicited feeding following microinjection into the NAcc [1– 3,10,30,37,70,71] suggested that the m opioid receptor was implicated in the mediation of opioid-induced feeding within the NAcc. This hypothesis was supported by the observation that b-FNA, a m-selective opioid receptor antagonist that alkylates this receptor [44] significantly and dose-dependently reduced DAMGO-induced feeding in the NAcc. The m opioid receptor has been subdivided into high-affinity m 1 and lower-affinity m 2 opioid binding sites (see review in Ref. [41]) which can be pharmacologically differentiated using the selective m 1 opioid antagonist, naloxonazine [13]. The respective ability of b-FNA (m 1 1 m 2 ) and the inability of naloxonazine (m 1 ) to alter DAMGO-induced feeding in the NAcc strongly suggest that the m 2 opioid receptor subtype mediates DAMGO-induced feeding in the NAcc, a pattern that was also observed for

selective m antagonist effects in the NAcc for feeding under deprivation, glucoprivic and palatable conditions [5,21]. Differentiations between m 1 -mediated and m 2 -mediated effects upon other forms of opioid-induced feeding following ventricular administration of opioid antagonists have been described as well (e.g. see review in Ref. [4]). Although DAMGO-induced feeding in the NAcc was largely unaffected by NAcc pretreatment with the selective d 1 -opioid antagonist, DALCE [7], this response was significantly reduced by the higher, but not lower dose of the selective d 2 -opioid antagonist, NTII [46] in the NAcc. Further, NAcc pretreatment with the higher, but not lower dose of the k 1 opioid antagonist, NBNI [45] significantly reduced DAMGO-induced feeding in the NAcc as well. This latter effect is quite similar to the abilities of ventricular administration of the same m and k 1 opioid antagonists to reduce feeding induced by ventricular administration of DAMGO [26,27]. Three explanations can account for these results: (a) DAMGO is not a selective m opioid agonist in feeding studies; (b) the antagonists are

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Fig. 5. Alterations in spontaneous food intake (g, 6S.E.M.) following a 5-mg dose of the d 1 -selective opioid agonist, DPDPE (DPD) bilaterally in the NAcc shell relative to control treatment. The upper and middle panels depict effects upon DPDPE-induced feeding following pretreatment (24 h) with different doses (mg) of either the selective d 1 -opioid antagonist, DALCE or the selective d 2 -opioid antagonist, naltrindole isothiocyanate (NTII), respectively. The lower panel depicts effects upon DPDPE-induced feeding following pretreatment (1 h) with different doses (mg) of the selective k 1 -opioid antagonist, nor-binaltorphamine (NBNI).

not entirely selective for their respective opioid receptor subtypes; and / or (c) feeding elicited by m opioid agonists in the NAcc is mediated through multiple opioid receptors. The first of these explanations is quite unlikely given the convergence of evidence using antisense oligodeoxynucleotide (AS ODN) probes directed against either the MOR-1, KOR-1 or DOR-1 opioid receptor clones (see reviews in Refs. [40,50,65]). DAMGO-induced feeding is

selectively reduced following ventricular pretreatment with AS ODN probes directed against either exons 1 or 4 of the MOR-1 clone, but unaffected by probes directed against either exons 2 or 3 of the MOR-1 clone or a missense probe [25]. These AS ODN effects upon DAMGO-induced feeding were comparable in magnitude to those reductions observed following b-FNA administration [25]. In contrast, AS ODN probes directed against the MOR-1 clone

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Fig. 6. Alterations in spontaneous food intake (g, 6S.E.M.) following a 5-mg dose of the d 2 -selective opioid agonist, Deltorphin (Delt) bilaterally in the NAcc shell relative to control treatment. The upper and middle panels depict effects upon Delt-induced feeding following pretreatment (24 h) with different doses (mg) of either the selective d 2 -opioid antagonist, NTII or the selective m-opioid antagonist, b-FNA, respectively. The lower panel depicts effects upon DAMGO-induced feeding following pretreatment (1 h) with different doses (mg) of the selective k 1 -opioid antagonist, nor-binaltorphamine (NBNI).

that significantly reduced DAMGO-induced feeding failed to affect feeding induced by ventricular administration of either the d 2 opioid agonist, Deltorphin or the k 1 opioid agonist, U50488H [24]. Although NBNI was initially proposed as a selective and reversible k 1 opioid antagonist [45], both characteristics have been called into question such that the antagonist has shown some longer-acting behavioral and biochemical effects [15,20,39]. Further,

chronic, but not acute administration of NBNI appears to act at multiple opioid receptor subtypes rather than selectively at the k 1 site [56]. In contrast, the selectivity of NTII as a d 2 opioid antagonist [46] has not been open to as many questions. The lack of further opioid subtype receptor antagonists, particularly for the k 1 receptor, precludes further and definitive rectification of this issue. Even if the third hypothesis indicating that DAMGO-

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induced feeding in the NAcc is mediated through multiple opioid receptors in this structure is true, the potency and magnitude of these effects strongly suggest that the m receptor, and particularly its low-affinity m 2 opioid binding site play more critical roles in this response than either the d 2 or k 1 opioid receptor subtypes. Both DPDPE (d 1 ) and Deltorphin (d 2 ) opioid agonists in the NAcc produced comparable magnitudes and durations of feeding, confirming previous suggestions that the d receptor is involved in agonist-induced feeding in this structure [1,71]. Feeding elicited by DPDPE in the NAcc was reduced by NAcc pretreatment with either m, d 1 , d 2 or k 1 opioid receptor subtype antagonists, but not by m 1 opioid antagonism. Indeed, it appeared that m receptor antagonism with b-FNA produced more pronounced effects upon DPDPE-induced feeding than either DALCE, NTII or NBNI. Therefore, unlike ventricular analgesic studies in which DPDPE-induced analgesia is selectively reduced by pretreatment with DALCE, but not NTII [18,19,55], it appears that multiple d receptor subtypes mediate the ingestive response elicited by DPDPE within the NAcc. Indeed, ventricular administration of DPDPE elicits feeding which was significantly reduced by the general opioid antagonist, naltrexone and the d 2 opioid antagonist, NTII, but not by the dose range (20–40 mg) of the d 1 opioid antagonist, DALCE employed in this study [69]. In the present study, doses of 1–4 mg of DALCE prevented the full expression of DPDPE-induced feeding in the NAcc; both solubility and availability issues precluded us from testing higher doses of this antagonist in the NAcc. The putative d 2 opioid receptor subtype agonist, Deltorphin, displayed an entirely different pattern of sensitivity to multiple opioid receptor subtype antagonists. Deltorphin-induced feeding was unaffected by NAcc pretreatment of the d 2 opioid antagonist, NTII, even at doses as high as 8 mg, the limit of solubility. The inability of NTII to affect Deltorphin-induced feeding in the NAcc could not be attributed to any perturbation in antagonist efficacy since NAcc NTII pretreatment concomitantly reduced feeding elicited by either DAMGO or DPDPE in the NAcc. Previous work [69] indicated that ventricular administration of Deltorphin produced feeding that was sensitive to both d 1 and d 2 antagonism; unanticipated unavailability of the antagonist, DALCE precluded testing upon Deltorphin-induced feeding within the NAcc. Further support for the ability of d receptor antagonists to reduce Deltorphin-induced feeding following ventricular administration [69] has been provided by the ability of AS ODN probes directed against the DOR-1 clone to significantly reduce Deltorphin-induced feeding [24]. Indeed, it has been proposed in analgesic AS ODN studies that the DOR-1 clone possesses more similarities with the pharmacologically-described d 2 opioid receptor subtype [52]. Therefore, it appears that the inability of NTII to alter Deltorphin-induced feeding in the NAcc as expected is probably due to the ineffectiveness of delivering high

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enough antagonist doses into the structure rather than to the failure of Deltorphin to act at a d 2 receptor in this site and paradigm. In marked contrast to the respective abilities of b-FNA and NBNI to reduce feeding elicited by either DAMGO or DPDPE in the NAcc, pretreatment with each of these antagonists significantly and dose-dependently enhanced Deltorphin-induced feeding in the NAcc. Thus, while DPDPE and DAMGO share some common opioid receptor substrates in mediating their ingestive responses in the NAcc, the feeding response induced by Deltorphin appears to activate different pathways in the NAcc that are modulated differentially by opioid receptor subtypes. As indicated previously, m, d and k receptors have been localized in the NAcc using receptor autoradiographic and mRNA gene expression techniques [14,28,32,33]. While these techniques verify the presence of these receptors within the nucleus, they cannot specify the synaptic arrangement of these receptors relative to each other and to other transmitter and modulator systems within the nucleus. It is known that proenkephalin and prodynorphin mRNA are co-localized with dopamine and substance P neurons [66,67] and with D 1 , but not D 2 dopamine receptors [8]. Further, the DOR-1 opioid receptor clone appears to be on axon terminals apposed to terminals immunoreactive to the dopamine transporter [61]. Moreover, agonists at m and d 2 receptors stimulate dopamine release in the NAcc [29,43,68]. We [49] have recently examined the roles of D 1 and D 2 receptor antagonist pretreatment in the NAcc upon feeding elicited by DAMGO and Deltorphin. Other ultrastructural studies have suggested that dynorphin activation of k opioid receptors, Met 5 -enkephalin activation of d opioid receptors, and Leu 5 -enkephalin activation of m opioid receptors in the NAcc may act on spiny neurons modulating responses to excitatory and inhibitory amino acids [60,62,63]. Further, co-localization in the shell region of the NAcc have been observed for both m and NMDA receptors [12] as well as for m opioid receptors and GABA-containing neurons [64]. Studies [9,72] are underway examining the relationship between DAMGO-induced feeding in the NAcc and the ability of either excitatory [31,59] or inhibitory [57,58] amino acid agonists and antagonists to regulate feeding in this nucleus. Taken together, regulation of the NAcc by these multiple transmitter systems appear to play an integral role in the elucidation of ingestive and other (e.g. see reviews in Refs. [16,53]) motivated behaviors, and ingestion mediated by m and d opioid receptor agonists appear to employ multiple opioid receptors within the NAcc in the differential expression of this ingestive response.

Acknowledgements This research was supported by National Science Foundation Grant IBN 98-16699 to RJB. MM was a CUNY Project ASCEND Fellow.

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