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Analysis of opioid receptor subtype antagonist effects upon mu opioid agonist-induced feeding elicited from the ventral tegmental area of rats
Nicole Lamonte, Joyce A. Echo, Tsippa F. Ackerman, Garrison Christian, 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 14 November 2001

Abstract The present study examined opioid receptor(s) mediation of feeding elicited by mu opioid agonists in the ventral tegmental area using general or selective opioid antagonist pretreatment. Naltrexone as well as equimolar doses of selective mu and kappa, but not delta opioid antagonists in the ventral tegmental area signicantly reduced mu agonist-induced feeding, indicating a pivotal role for these receptor subtypes in the full expression of this response. 2002 Elsevier Science B.V. All rights reserved.
Theme: Neurotransmitters, modulators, transporters and receptors Topic: Opioids: anatomy, physiology and behavior Keywords: Feeding; DAMGO; Ventral tegmental area; Opioid antagonist; Mu opioid receptor; Kappa opioid receptor; Delta opioid receptor

The reciprocal pathways between the ventral tegmental area (VTA) and nucleus accumbens (Nacc) have been implicated in the mediation of reward and reinforcement related to ingestive and drug-seeking behaviors, including those induced by opiates (see reviews: [8,2224]). Thus, food intake is stimulated following intracranial administration of opioid agonists directly into either the Nacc [2,3,13,14] or the VTA [6,7,14,15,17,18]. These studies implicated mu and secondarily delta, but not kappa opioid receptors in this response. Administration of the selective mu opioid agonist, [D-Ala 2 , NMe-Phe 4 , Gly-ol 5 ]-enkephalin (DAMGO), into the VTA stimulates food intake and related gnawing behaviors under both spontaneous and deprived conditions [1,17,18], presumably by increasing dopamine transmission in the Nacc and prefrontal cortex [16]. However, administration of general and selective opioid antagonists into either the Nacc or VTA produce differential effects upon feeding responses elicited by regulatory challenges or exposure to palatable substances. Whereas naltrexone potently and robustly reduced intake
* Corresponding author. Tel.: 11-718-997-3543; fax: 11-718-9973257. E-mail address: richard bodnar@qc.edu (R.J. Bodnar). ]

under deprivation, glucoprivic and palatable conditions following administration into the Nacc [5,9], only high doses of naltrexone administered into the VTA produced marginal effects upon these ingestive responses [21]. Further, the use of selective opioid receptor subtype antagonists revealed a marked role for mu, and secondarily kappa opioid receptors in mediating these responses in the Nacc [5,9], but only a marginal role for delta opioid receptors in mediating these responses in the VTA [21]. In contrast to receptor-selective blockade of opioid receptor subtype agonist effects upon analgesia by corresponding opioid receptor subtype antagonists (e.g. Refs. [4,19]), opioid antagonist effects upon opioid agonistinduced feeding do not display this clear-cut specicity. Thus, feeding responses elicited by ventricular administration of selective mu or delta opioid agonists are blocked by pretreatment with either selective mu or kappa opioid antagonists [11,12]. Moreover, feeding responses elicited by either selective mu or delta 1 opioid agonists administered into the Nacc are blocked by pretreatment with either mu, delta 1 , delta 2 or kappa 1 opioid antagonists [20]. The interconnections between the Nacc and VTA, and the ability of both nuclei to sustain feeding responses following intracranial administration of selective mu and delta

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opioid agonists would suggest that the VTA should display a similar pattern of antagonist sensitivity. In contrast, the differential opioid antagonist effects in both nuclei upon feeding under challenge and palatable conditions would alternatively suggest a different pattern of effects in the VTA relative to the Nacc. To assess these possibilities, the present study examined whether feeding responses elicited by the selective mu opioid agonist, DAMGO, in the VTA were differentially sensitive to a dose range of the general opioid antagonist naltrexone following VTA or systemic administration, as well as VTA pretreatment with an equimolar dose range of selective mu (beta-funaltrexamine), kappa 1 (nor-binaltorphamine) or delta (naltrindole) opioid receptor subtype antagonists. Adult male albino SpragueDawley rats (Charles River Laboratories, Kingston, NY, 80100 days of age) were individually housed in wire-mesh cages and maintained on a 12-h light / 12-h dark cycle with Purina rat chow and water available ad libitum. Bilateral stainless steel guide cannulae (26 gauge, Plastics One Inc., Roanoke, VA) were aimed stereotaxically (Kopf Instruments, Tujunda, CA) at the VTA using the following coordinates: incisor bar ( 15 mm), 5.6 mm posterior to the bregma suture, 62.2 mm lateral and angled 108 towards the sagittal suture, and 8.4 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 to recover for at least 2 weeks before behavioral testing began. In all experiments, each of the rats was initially tested for spontaneous food intake over a 4-h time course at 310 h into the light cycle during which minimal amounts of intake are typically observed. Cumulative intakes were assessed 1, 2 and 4 h following the placement of preweighed food pellets onto the oor of the cage to allow easy access to the food. All intakes were adjusted for spillage, which was collected by paper towels placed under the wire-mesh cages. Rats were tested over at least two baseline sessions to establish consistently low intakes, and minimize the occurrence of novelty-induced feeding. In assessing whether VTA pretreatment with the general opioid antagonist, Ntx altered DAMGO-induced feeding elicited from the VTA, the rst group of rats received the following bilateral microinjection conditions: (a) vehicle (n 521), (b) DAMGO (5.0 mg, 2.5 mg each side, n 521, Peninsula Laboratories, Belmont, CA), and total Ntx (SigmaAldrich, St Louis, MO) doses of (c) 5 (2.5 mg each side, n 57), (d) 20 (10 mg each side, n 57), (e) 50 (25 mg each side, n 57) or (f) 100 (50 mg each side, n 57) mg administered 20 min prior to DAMGO (5 mg). In assessing whether systemic pretreatment with Ntx altered DAMGOinduced feeding elicited from the VTA, a second separate group of rats received the following conditions: (a) vehicle (n 512), (b) DAMGO (VTA: 5 mg, n 512), and subcutaneous Ntx doses of (c) 0.1 (n 56), (d) 1 (n 56), or (e) 2 (n 56) mg / kg administered 20 min prior to VTA DAMGO

(5 mg). In assessing whether VTA pretreatment with the mu opioid antagonist, BFNA altered DAMGO-induced feeding elicited from the VTA, a third separate group of rats received the following bilateral microinjection conditions: (a) vehicle (n 512), (b) DAMGO (5 mg, n 512), and total B-FNA (Research Biochemicals Int., Natick, MA) doses of (c) 0.4 (0.8 nmol, 0.2 mg each side, n 57) and (d) 4 (8.2 nmol, 2 mg each side, n 59) mg administered 24 h prior to VTA DAMGO (5 mg). In assessing whether VTA pretreatment with the kappa opioid antagonist, NBNI altered DAMGO-induced feeding elicited from the VTA, a fourth separate group of rats received the following bilateral microinjection conditions: (a) vehicle (n 59), (b) DAMGO (5 mg, n 59), and total NBNI (Research Biochemicals Int., Natick, MA) doses of (c) 0.6 (0.8 nmol, 0.3 mg each side, n 57) and (d) 6 (8.2 nmol, 3 mg each side, n 57) mg administered 20 min prior to DAMGO (5 mg). In assessing whether VTA pretreatment with the delta opioid antagonist, NTI altered DAMGOinduced feeding elicited from the VTA, a fth separate group of rats received the following bilateral microinjection conditions: (a) vehicle (n 510), (b) DAMGO (5 mg, n 510), and total NTI (Research Biochemicals Int., Natick, MA) doses of (c) 0.4 (0.8 nmol, 0.2 mg each side, n 57) and (d) 4 (8.2 nmol, 2 mg each side, n 57) mg administered 20 min prior to DAMGO (5 mg). In all studies, cumulative intakes were assessed 1, 2, and 4 h following the last injection. The pretreatment intervals were chosen to optimize specic opioid receptor antagonism. All drugs, dissolved in distilled water, were administered bilaterally in 1-ml volumes through a stainless steel internal cannula (33 gauge) connected to a Hamilton microsyringe by polyethylene tubing. A minimum of 1 week elapsed between treatment conditions in each group to minimize antagonist-induced carryover effects. At the completion of testing, all animals received an overdose of Euthansol (Del Marva Laboratories, 390 mg / ml sodium pentobarbital; 50 mg / ml sodium phenytoin; 0.05 ml / kg). Transcardiac perfusions were performed with 0.9% normal saline followed by 10% buffered formalin. Sections were examined by an observer unfamiliar with the behavioral data; only animals with conrmed cannula placements were included in the experimental groups. All of the animals included in this study had proper cannulae placements within the area of the VTA, bordered as far rostrally as the level of the fasciculus retroexus and commissure of the superior colliculus, bordered as far caudally as the level of the interpeduncular nucleus, and bordered as far dorsally as the caudal linear nucleus of the raphe. There were several animals with misplaced cannulae that failed to display DAMGO-induced feeding, and therefore, were never used in any antagonistagonist condition or in any data analysis. Separate repeated-measures one-way analyses of variance were performed at each cumulative intake point. Tukey corrected comparisons (P ,0.05) were used to

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assess signicant alterations induced by agonist administration relative to vehicle treatment, and signicant alterations induced by antagonist pretreatment upon agonistinduced feeding relative to DAMGO treatment. Analyses revealed inconsistent signicant ingestive responses to DAMGO after 1 h across the ve groups; therefore, food intake after 2 and 4 h was only evaluated in each paradigm. VTA pretreatment with Ntx signicantly and dose-dependently reduced DAMGO-induced feeding elicited from the VTA after 2 (F(5,100)548.49, P ,0.0001) and 4 (F 5 63.77, P ,0.0001) h (Fig. 1, upper panel). The signicant increases in DAMGO-induced feeding after 2 and 4 h relative to vehicle treatment were signicantly reduced by the two highest (50 [85100% reduction] and 100 [100% reduction] mg), but not the two lower (5 and 20 mg) Ntx doses in the VTA. Systemic pretreatment with Ntx also signicantly and dose-dependently reduced DAMGO-induced feeding elicited from the VTA after 2 (F(4,44)5 6.90, P ,0.0002) and 4 (F 513.19, P ,0.0001) h (Fig. 1, lower panel). Pretreatment with the 1 and 2 mg / kg doses of Ntx prevented the occurrence of the signicant increases in DAMGO-induced feeding after 2 (100% reduction) and 4 (5359% reduction) h; the lower (0.1 mg / kg) Ntx dose was effective after 2, but not 4 h. These data indicate that an opioid receptor in the VTA is necessary for the full expression of DAMGO-induced feeding elicited from the VTA. VTA pretreatment with the mu opioid antagonist, BFNA, produced signicant dose-dependent and time-dependent reductions of DAMGO-induced feeding elicited from the VTA after 2 (F(3,33)517.33, P ,0.0001) and 4 (F 522.73, P ,0.0001) h (Fig. 2, upper panel). Whereas all groups displayed signicant increases in intake after 2 h relative to vehicle treatment, VTA pretreatment with the 0.4 (85% reduction) and 4 (53% reduction) mg doses of BFNA signicantly reduced the magnitude of DAMGOinduced feeding elicited from the VTA after 4 h, indicating a role for mu opioid receptors in the full expression of DAMGO-induced feeding elicited from the VTA. VTA pretreatment with the kappa opioid antagonist, NBNI signicantly and dose-dependently reduced DAMGO-induced feeding elicited from the VTA after 2 (F(3,24)5 8.17, P ,0.0001) and 4 (F 512.24, P ,0.0001) h (Fig. 2, middle panel). The signicant increases in DAMGO-induced feeding after 2 and 4 h relative to vehicle treatment were signicantly reduced by the higher (6 mg, 7392% reduction), but not lower (0.6 mg) NBNI doses in the VTA, indicating a role for kappa opioid receptors in the full expression of DAMGO-induced feeding elicited from the VTA. DAMGO in the VTA produced signicant increases in food intake after 2 (F(3,27)59.67, P ,0.0002) and 4 (F 517.35, P ,0.0001) h relative to vehicle treatment following the agonist alone and when it was paired with VTA pretreatment with both doses of the delta opioid antagonist, NTI (Fig. 2, lower panel), suggesting that this

Fig. 1. Alterations in food intake (mean6S.E.M.) 2 and 4 h following microinjection of the mu opioid agonist, DAMGO (DAM: 5 mg) in the ventral tegmental area (VTA) in animals pretreated with the general opioid antagonist, naltrexone (Ntx) administered either into the VTA (Panel A: mg doses) or subcutaneously (Panel B: mg / kg doses). The asterisks (*) in this and the subsequent gure denote signicant increases in agonist-induced feeding relative to vehicle treatment (Tukey corrected comparison, P ,0.05). The crosses ( 1 ) in this and the subsequent gure denote signicant decreases following antagonist pretreatment relative to agonist treatment alone (Tukey corrected comparison, P ,0.05).

receptor subtype fails to play a role in the full expression of DAMGO-induced feeding elicited from the VTA. Given the reciprocal interconnections between the VTA and Nacc as well as their ability to support robust feeding responses in rats following administration of the mu-opioid receptor agonists such as DAMGO, it was hypothesized

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Fig. 2. Alterations in food intake (mean6S.E.M.) 2 and 4 h following VTA microinjection of the mu opioid agonist, DAMGO (DAM: 5 mg) in animals receiving VTA pretreatment with equimolar ( mg) doses of either selective mu (Panel A: beta-funaltrexamine: BFNA), kappa (Panel B: nor-binaltorphamine: NBNI) or delta (Panel C: naltrindole: NTI) opioid antagonists.

that DAMGO-induced feeding elicited from the VTA should be similarly sensitive to naltrexone pretreatment as was observed in the Nacc shell. Systemic pretreatment with naltrexone dose-dependently reduced DAMGO-induced feeding elicited from the VTA, indicating that an opioid synapse is involved in the mediation of this response. However, when naltrexone pretreatment occurred within the VTA, the antagonist signicantly reduced VTA DAMGO-induced feeding at doses (50100 mg) that consistently reduced intake under deprivation, glucoprivic, and palatable conditions [21]. Lower (|520 mg) naltrexone doses reduced these same ingestive responses following microinjection into either the Nacc [5,9] or the hypothalamic paraventricular nucleus [10]. Furthermore, in agreement with previous work [1], qualitative behavioral observation of the animals revealed that VTA DAMGO at a dose of 5 mg produced such oral behaviors as biting the bottom of the wire mesh cage. These behaviors occurred shortly after DAMGO administration, dissipated quickly over time, and were not noted when animals were pretreated with opioid receptor antagonists. These anecdotal data suggest that feeding and these other adjunctive behaviors elicited by VTA DAMGO are mediated through opioid receptors. In contrast to receptor-selective opioid antagonist effects upon opioid agonist-induced analgesic responses (e.g. Refs. [4,19]), opioid antagonist effects upon opioid agonist-induced feeding have often involved multiple opioid receptor subtypes (e.g. Refs. [11,12,20]). Within the Nacc shell, DAMGO-induced feeding was signicantly reduced by selective mu, kappa and delta opioid receptor antagonists [20], and the present study examined whether this feeding response elicited from the VTA was altered by VTA pretreatment with an equimolar dose range of selective mu (beta-funaltrexamine), kappa 1 (nor-binaltorphamine) or delta (naltrindole) opioid receptor subtype antagonists. Consistent with a receptor-selective antagonist effect upon agonist-induced feeding, BFNA pretreatment in the VTA signicantly decreased VTA DAMGO-induced feeding after 4 h, indicating a role for mu opioid receptors in this response. Interestingly, pretreatment with kappa (NBNI), but not delta (naltrindole) opioid antagonists in the VTA signicantly and dose-dependently decreased DAMGO-induced feeding. The kappa-mediated effect occurred at a dose range equimolar to that of BFNA, indicating approximately equipotent antagonist actions. Therefore, DAMGO-induced feeding elicited from the VTA is similar to its parallel ingestive response elicited from the Nacc shell [20] in its dependence upon both intrinsic mu and kappa opioid receptors, but differs from the Nacc shell in its insensitivity to delta opioid antagonism. The fact that delta opioid antagonism failed to affect DAMGO-induced feeding elicited from the VTA differs markedly from the ability of delta opioid antagonism in the VTA to produce small, but signicant antagonism of feeding elicited by either deprivation, glucoprivation or

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N. Lamonte et al. / Brain Research 929 (2002) 96 100 [10] J.E. Koch, M.J. Glass, M.L. Cooper, R.J. Bodnar, Alterations in deprivation, glucoprivic and sucrose intake following general, mu and kappa opioid antagonists in the hypothalamic paraventricular nucleus of rats, Neuroscience 66 (1995) 951957. [11] A.S. Levine, M. Grace, C.J. Billington, B-funaltrexamine (B-FNA) decreases deprivation and opioid-induced feeding, Brain Res. 562 (1991) 281284. [12] A.S. Levine, M. Grace, C.J. Billington, P.S. Portoghese, Nor-binaltorphamine decreases deprivation and opioid-induced feeding, Brain Res. 534 (1990) 6064. [13] N.H. Majeed, B. Przewlocka, K. Wedzony, R. Przewlocki, Stimulation of food intake following opioid microinjection into the nucleus accumbens septi in rats, Peptides 7 (1986) 711716. [14] R.F. Mucha, S.D. Iversen, Increased food intake after opioid microinjections into nucleus accumbens and ventral tegmental area of rat, Brain Res. 397 (1986) 214224. [15] P. Nencini, J. Stewart, Chronic systemic administration of amphetamine increases food intake to morphine, but not to U50488H, microinjected into the ventral tegmental area in rats, Brain Res. 527 (1990) 254258. [16] M.B. Noel, A. Gratton, Electrochemical evidence of increased dopamine transmission in prefrontal cortex and nucleus accumbens elicited by ventral tegmental mu receptor activation in freely behaving rats, Synapse 21 (1995) 110122. [17] M.B. Noel, R.A. Wise, Ventral tegmental injections of morphine but not U50488H enhance feeding in food-deprived rats, Brain Res. 632 (1993) 6873. [18] M.B. Noel, R.A. Wise, Ventral tegmental injections of a selective mu or delta opioid enhance feeding in food-deprived rats, Brain Res. 673 (1995) 304312. [19] G.W. Pasternak, Pharmacological mechanisms of opioid analgesics, Clin. Neuropharmacol. 16 (1993) 118. [20] A. Ragnauth, M. Moroz, R.J. Bodnar, Multiple opioid receptors mediate feeding elicited by mu and delta opioid receptor subtype agonists in the nucleus accumbens shell in rats, Brain Res. 876 (2000) 7687. [21] A. Ragnauth, H. Ruegg, R.J. Bodnar, Evaluation of opioid receptor subtype antagonist effects in the ventral tegmental area upon food intake under deprivation, glucoprivic and palatable conditions, Brain Res. 767 (1997) 816. [22] J.D. Salamone, The involvement of nucleus accumbens dopamine in appetitive and aversive motivation, Behav. Brain Res. 61 (1994) 117133. [23] D.W. Self, L. Stein, Receptor subtypes in opioid and stimulant reward, Pharmacol. Toxicol. 70 (1992) 8794. [24] R.A. Wise, M.A. Bozarth, A psychomotor stimulant theory of addiction, Psychol. Rev. 94 (1987) 469492.

exposure to a palatable substance [21]. These data suggest strongly that the pattern of opioid receptor subtypes mediating particular opioid agonist-induced feeding responses is site-specic, and therefore careful analyses of intracerebral mapping studies with both selective opioid antagonists and agonists is required to reveal the complex circuitry by which multiple endogenous opioid systems mediate this and other motivational behaviors.

Acknowledgements This research was supported by National Science Foundation Grant IBN 98-16699 to R.J.B.

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