Endogenous opioids encode relative taste preference

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1 European Journal of Neuroscience, Vol. 24, pp , 2006 doi: /j x Endogenous opioids encode relative taste preference Sharif A. Taha, 1,2 Ebba Norsted, 1,2 Lillian S. Lee, 1,2 Penelope D. Lang, 1,2 Brian S. Lee, 1,2 Joshua D. Woolley 1,2 and Howard L. Fields 1,2 1 Ernest Gallo Clinic and Research Center, University of California, San Francisco, Emeryville, CA 94608, USA 2 Departments of Neurology and Physiology, and the Wheeler Center for the Neurobiology of Addiction, University of California, San Francisco, San Francisco, CA 94143, USA Keywords: food intake, nucleus accumbens, opioids, palatability, rat, ventral tegmental area Abstract Endogenous opioid signaling contributes to the neural control of food intake. Opioid signaling is thought to regulate palatability, the reward value of a food item as determined by orosensory cues such as taste and texture. The reward value of a food reflects not only these sensory properties but also the relative value of competing food choices. In the present experiment, we used a consummatory contrast paradigm to manipulate the relative value of a sucrose solution for two groups of rats. Systemic injection of the nonspecific opioid antagonist naltrexone suppressed sucrose intake; for both groups, however, this suppression was selective, occurring only for the relatively more valuable sucrose solution. Our results indicate that endogenous opioid signaling contributes to the encoding of relative reward value. Introduction A rich network of interrelated factors control feeding (Schwartz et al., 2000). These include the status of energy reserves, metabolic demand and the palatability of available foods. Of these, palatability is of particular interest because it plays an important role in driving caloric consumption in excess of metabolic demand, promoting obesity (Raynor & Epstein, 2001). Rats offered ad libitum access to a varied and palatable cafeteria diet ingest many more calories than animals consuming nutritionally balanced but bland rat chow; indeed, cafeteria diets offer a well established paradigm for experimentally inducing obesity (Rogers & Blundell, 1984). Sensory attributes of palatable foods are generally those which signal high energy density, such as sweetness or fatty texture. Opioid signaling in the central nervous system plays a major role in neural processing related to palatability. Opioid agonists such as morphine potentiate food intake selectively, with the largest effects seen in consumption of palatable sweet or high-fat foods (Evans & Vaccarino, 1990). Nonspecific opioid antagonists such as naltrexone and naloxone suppress food intake, again with the largest effects evident during consumption of highly palatable food items (Le Magnen et al., 1980). These effects do not occur through modulation of postingestive feedback, as they are apparent even in sham-feeding rats (Rockwood & Reid, 1982). In addition, morphine administration increases positive facial reactivity displays, measures which are tightly correlated with food preference (Pecina & Berridge, 1995). These findings suggest a specific role for opioids in signaling the reinforcing value of palatable food items. The nucleus accumbens (NAcc) is a critical site of opioid signaling related to palatability. Infusion of naltrexone directly into the NAcc causes a selective decrease in sated rats sucrose intake, with minimal effects upon consumption of (less preferred) chow (Kelley et al., Correspondence: Dr Sharif A. Taha, 1 Ernest Gallo Clinic and Research Center, as above. staha@phy.ucsf.edu Received 30 June 2005, revised 6 June 2006, accepted 8 June ). Conversely, infusion of the mu-opioid receptor (MOR) specific agonist [D-Ala2,N-Me-Phe4-Gly5-ol]-enkephalin (DAMGO) selectively increases intake of a variety of preferred substances, including sucrose, noncaloric saccharin and a dilute saline solution (Zhang & Kelley, 2002). These findings demonstrate an important role for NAcc opioid signaling in promoting consummatory behaviours related to preferred, highly palatable food items. The intrinsic sensory qualities of a particular food item are important determinants of its reward value, as measured by the food s ability to motivate appetitive behaviour and to sustain consumption. In addition, when different foods are available, food-directed behaviours (both appetitive and consummatory) are typically guided by the relative value of competing food choices. A food item which is avidly consumed under normal conditions, for instance, may be rejected when more preferable alternatives are available (Brosnan & De Waal, 2003). Consummatory contrast paradigms provide well established behavioural models for manipulating the relative value of food rewards such as sucrose solutions (Flaherty, 1982). In anticipatory contrast paradigms, consumption of a palatable solution is suppressed by subsequent presentation of a second, more reinforcing, solution. This contrast effect is thought to develop through learned anticipation of the second solution (Flaherty & Checke, 1982; Flaherty & Rowan, 1985), and is dependent on the relative reward value of the contrasted solutions (Flaherty et al., 1994). We recently reported that a subset of NAcc neurons encode information related to the changing relative value of a sucrose solution modulated using a contrast paradigm (Taha & Fields, 2005). Because MOR agonists microinjected into the NAcc can increase consumption of palatable foods, we hypothesized that endogenous opioids contribute to this encoding and to consummatory behaviours guided by relative value. In the present study, an anticipatory contrast paradigm was used to modulate the relative value of an otherwise identical 4% sucrose solution in two groups of rats. Systemic administration of the nonspecific opioid antagonist naltrexone selectively reduced consumption of the relatively more palatable sucrose

2 Opioids encode relative value 1221 solution for each group, demonstrating that endogenous opioids participate in signaling learned relative value. Materials and methods All procedures used were approved by the University of California, San Francisco, Animal Care and Use Committee. Male Long-Evans rats ( g) were randomly assigned to one of two experimental groups (4-0 or 4-20 group; see below) upon arrival in the animal care facility. Rats were habituated to handling for 1 week, then, for those included in the NAcc or ventral tegmental area (VTA) microinfusion studies, surgically implanted with bilateral cannulae prior to training in the anticipatory contrast paradigm. Rats were allowed ad libitum access to food and water at all times throughout the experiments. Training took place daily in operant chambers (Medical Associates, Georgia, VT, USA) equipped with a single lick spout with attached photobeam lickometer. Sessions lasted 30 min and consisted of two successive 15-min periods. During the first 15 min of each session, rats in both the 4-0 group and the 4-20 group received free access to an identical 4% sucrose solution. During the second 15-min period, rats in the 4-0 group received free access to a 0% sucrose solution. Rats in the 4-20 group received free access to a 20% sucrose solution (Fig. 1A). The number of licks delivered to the spout was recorded in 1-min bins throughout experimental sessions. Each lick resulted in consumption of 5 ll solution (volume consumed per lick was calculated in preliminary experiments by recording licks while rats consumed a premeasured volume of 4% sucrose). A masking white noise cue (90 db) was present throughout training and experimental sessions. Daily training continued until the mean consumption of 4% sucrose in the 4-0 group reached 2500 licks. This threshold value allowed sufficient time for a robust contrast effect (a large difference in 4% sucrose consumption between the two groups) to emerge. Training time averaged 24 ± 2 days across all animals. For cannula implantation, surgical anaesthesia was induced with ketamine and xylazine (50 10 mg kg) and maintained with isofluorane (3% in O 2 ). Cannulae were directed at the NAcc (target coordinates from bregma: anterioposterior, +1.0; lateral, ±1.1; ventral, 7.5 mm) or the VTA (anterioposterior, )5.2; lateral, ±0.5; ventral, 8.0 mm). Cannulae were implanted 2 mm above the target coordinates; injectors projected 2 mm beyond the end of the cannulae tips. Endogenous opioid encoding of relative value was tested with administration of the nonspecific opioid antagonist naltrexone. All naltrexone treatments (both systemic and intracranial routes of delivery) were administered 10 min before the beginning of experimental sessions. During the 10-min interval between drug administration and testing, rats were returned to their home cages, with food hoppers removed. Systemic naltrexone (1 mg kg) was delivered subcutaneously. A total of 45 rats were used in systemic naltrexone experiments (21 in the 4-0 group, 24 in the 4-20 group). Each rat was injected with naltrexone and control saline, with injections taking Fig. 1. Consummatory contrast paradigm. (A) All rats were allowed 15 min free access to a 4% sucrose solution. Immediately thereafter, one group of rats (4-0 group) was allowed 15 min free access to a 0% sucrose solution. The second group of rats (4-20 group) was allowed 15 min free access to a highly palatable 20% sucrose solution. Rats were trained daily using this paradigm for 3 weeks before testing drug effects. (B) The consummatory contrast paradigm created a marked difference in the two groups consumption of the 4% sucrose solution; 4% sucrose consumption averaged 2514 ± 154 licks in the 4-0 group (n ¼ 52 rats) but 1116 ± 126 licks in the 4-20 group (n ¼ 54 rats). Graphs show mean number of licks for each group in the absence of any drug treatment. *P < (C) Four per cent sucrose consumption in the first half of the experimental session diverged early in the session for 4-0 and 4-20 groups, and this difference was maintained throughout the session. (D) Twenty per cent sucrose consumption was robust in the second half of the experimental session in the 4-20 group, while 0% sucrose consumption remained low.

3 1222 S. A. Taha et al. place in a counterbalanced fashion, and a single rest day allowed between injections. In NAcc-cannulated rats, three doses of naltrexone (0.5 ll volume) were infused into each NAcc: 6.5, 20 and 40 lg; 49 rats were used in intra-nacc infusions (24 rats in the 4-0 group, 25 in the 4-20 group). Each rat received all treatments (one saline and three naltrexone infusions). Infusions were performed every other day, allowing a rest day in between injections, and the order of naltrexone and saline infusions was randomized across days. In VTA-cannulated rats the experimental design was identical, with three doses of naltrexone (0.5 ll volume) infused into each VTA: 1, 5 and 20 lg; 48 rats were used in intra-vta infusions (24 rats in the 4-0 group, 24 in the 4-20 group). Data illustrating the contrast effect (Fig. 1B and C) were drawn from a total of 106 drug-naïve rats on the final day of training in the contrast paradigm (52 in the 4-0 group, 54 in the 4-20 group); these rats were subsequently used for systemic or intracranial (NAcc or VTA) naltrexone administration experiments. Statistical analyses of the contrast paradigm and the effects of systemic naltrexone administration were performed using t-tests; when comparing effects within subjects, paired t-tests were used. To analyse drug effects following intracranial naltrexone infusion, statistical analyses were performed using one-way repeated-measures anovas, with drug concentration as the dependent measure. Finally, t-tests were used to compare naltrexone-induced suppression of 4% sucrose consumption in the 4-0 and 4-20 groups. Naltrexone-induced suppression was calculated for each rat as the difference in consumption following naltrexone administration relative to that following saline; these difference values were then compared across 4-0 and 4-20 groups. For the NAcc and VTA microinfusions, consumption after administration of the highest dose of naltrexone was used for this calculation. To analyse cannula placements, rats were deeply anaesthetized and cannula sites were marked by passing a 20-lA current for 20 s through an electrode cut to the same final length as the injectors. Rats were perfused with a solution of 10% formaldehyde and 3% potassium ferricyanide to mark sites of iron deposition. Brains were cryoprotected, and coronal sections cut and mounted. Cannula positions were located under a light microscope and recorded on atlas figures adapted from Paxinos & Watson (1997). Results The anticipatory contrast paradigm (Fig. 1A) created a robust difference between the two groups in the consumption of the 4% sucrose solution; 4% sucrose consumption in the 4-0 group averaged 2514 ± 154 (mean ± SEM) licks (Fig. 1B). In the 4-20 group consumption was 56% lower, averaging 1116 ± 126 licks. This reduction in total licking was highly significant (t 104 ¼ 7.0, P < 0.001). Within each group, consumption of different sucrose reinforcers was also significantly different. In the 4-0 group, consumption of 0% sucrose during the second half of the experimental session averaged 117 ± 47 licks, significantly less than occurred during 4% sucrose consumption in the first half of the session (t 51 ¼ 14.2, P < 0.001). In the 4-20 group, 20% sucrose consumption averaged 1893 ± 156 licks, significantly more than 4% sucrose consumption in the same group (t 53 ¼ 3.9, P < 0.001). It is notable that, in this 4-20 group, the volume of 20% sucrose consumed exceeded 4% sucrose consumption despite occurring in the second half of the session, when satiety effects are more likely to have suppressed overall intake. Figure 1C and D shows cumulative consumption (in licks) for both 4-0 and 4-20 groups for the first half (Fig. 1C) and second half (Fig. 1D) of the experimental paradigm to illustrate the time course of different consumption rates. Differences in 4% sucrose consumption between the two groups emerged rapidly and were maintained for the duration of the first 15-min consumption period (Fig. 1C). In the second half of the session, 20% sucrose consumption was robust while only small amounts of 0% sucrose were consumed (Fig. 1D). To test for a potential role of endogenous opioids in encoding relative value, we tested the effects of systemic naltrexone injection (1 mg kg) on sucrose intake in each of the two groups. Naltrexone caused a significant reduction in 4% sucrose consumption in the 4-0 group (Fig. 2A). Rats injected with a control saline solution averaged 2621 ± 206 licks. This declined to 1637 ± 123 licks following subcutaneous naltrexone administration (t 20 ¼ 4.6, P < 0.001). Naltrexone administration did not alter subsequent 0% sucrose consumption (Fig. 2B; t 20 ¼ 0.8, P ¼ 0.45). Administration of systemic naltrexone to the 4-20 group caused strikingly different results: 4% sucrose consumption remained robust, at levels very similar to those occurring following control saline injections. Consumption of 4% sucrose averaged 1047 ± 159 licks following naltrexone injection, relative to 1234 ± 203 licks following saline injection. This 15% reduction in consumption was not significant (Fig. 2C; t 23 ¼ 1.9, P ¼ 0.07). While naltrexone had negligible effects upon 4% sucrose consumption in this group, subsequent 20% sucrose consumption was substantially reduced; 20% sucrose consumption averaged 1336 ± 135 licks following control saline administration, but declined 51% to 656 ± 69 licks following systemic naltrexone administration (Fig. 2D; t 23 ¼ 5.6, P < 0.001). Importantly, this finding also demonstrates that the absence of a naltrexone effect upon 4% consumption in the 4-20 group was not the result of a floor effect. Under control conditions, 4% and 20% sucrose consumption were quite similar in this 4-20 group; however, despite these similar levels of baseline consumption, naltrexone significantly reduced consumption of the latter but not the former. Thus, while blocking endogenous opioid signaling selectively reduced intake of a relatively more reinforcing 4% sucrose solution in the 4-0 group, it did not alter consumption of a relatively less reinforcing 4% sucrose solution in the 4-20 group. Direct comparison of the magnitude of the naltrexone-induced reduction in consumption in the two groups showed that this was significantly larger in the 4-0 group than in the 4-20 group (Fig. 3; t 43 ¼ 3.5, P < 0.001). Despite the identical sensory properties of the 4% sucrose solution for the two groups, endogenous opioid signaling (and its consequent effects upon behaviour) were very different. In the 4-20 group of rats, consumption of the 20% sucrose solution was dramatically reduced by naltrexone administration. For each group, then, systemic naltrexone administration selectively reduced consumption of only the relatively most reinforcing solution. These results demonstrate that opioid signaling related to palatability flexibly encodes the relative value of a sucrose reinforcer. Opioid agonists and antagonists injected into the NAcc have effects similar to those seen after systemic routes of administration, suggesting that the NAcc is an important site of opioid signaling related to palatability. Therefore, we next studied the effects of direct injection of naltrexone into the NAcc to attempt to localize the critical site of endogenous opioid signaling of relative value. Naltrexone microinfusion in the NAcc did not have significant effects upon consumption in 4-0 or 4-20 groups. The largest effects were seen for 4% consumption in the 4-0 group (Fig. 4A), where naltrexone administration produced a trend toward significantly reduced consumption which did not reach significance (no effect of drug, F 3,69 ¼ 2.5, P ¼ 0.06). Consumption during all other conditions was unaffected by naltrexone administration (Fig. 4B D: all F < 1.3, all P >> 0.05). As a direct measure of naltrexone effects related to relative value, we examined the magnitude of the highest

4 Opioids encode relative value 1223 Fig. 2. Systemic naltrexone reduced 4% sucrose consumption in the 4-0 group, but not the 4-20 group. (A) Following saline injections, rats consumed 2621 ± 206 licks of 4% sucrose. Consumption was significantly reduced to 1637 ± 123 licks (37% reduction; P < 0.001) following subcutaneous administration of naltrexone (1 mg kg). The left panel shows the total number of licks delivered under each condition. The right panel shows cumulative licks as a function of time in the experimental session (shaded symbols, naltrexone administration; open symbols, saline). *P < for A D. (B) Subcutaneous administration of naltrexone did not significantly alter 0% sucrose consumption relative to saline control. Insets show the same data with the y-axis rescaled. (C) Four per cent sucrose consumption in the 4-20 group following saline (1234 ± 203) and naltrexone (1047 ± 159) administration were very similar. (D) Twenty per cent sucrose consumption was significantly reduced following naltrexone administration (656 ± 69) relative to saline injection (1336 ± 135; a 51% reduction). Fig. 3. Naltrexone-induced reduction in 4% sucrose consumption was larger in the 4-0 group than the 4-20 group. Box plots (indicating 25th, median and 75th percentile values) show naltrexone-induced reduction in 4% sucrose consumption relative to saline control. The magnitude of the median reduction in the 4-0 group was significantly larger than that occurring in the 4-20 group (*P <0.001). dose of naltrexone in reducing 4% sucrose consumption in the two groups. The reduction in 4% sucrose consumption in the 4-0 group caused by naltrexone (mean reduction of 355 ± 187 licks) trended toward a larger effect than that observed in the 4-20 group (mean increase of 37 ± 99 licks following naltrexone administration), but this difference did not reach significance (Fig. 4E; t 47 ¼ 1.9, P ¼ 0.07). Opioid modulation of food intake is not confined to the NAcc, but rather occurs in a number of other brain regions. Opioids acting in the VTA affect feeding behaviours, and an extensive literature implicates VTA neurons in encoding reward information (Schultz, 2001; Roitman et al., 2004). Therefore, we extended our study to include microinfusion of naltrexone directly into the VTA. Similar to results found for NAcc microinfusion, intra-vta naltrexone infusion had the largest effects upon 4% sucrose consumption in the 4-0 group (Fig. 5A; significant effect of drug, F 3,69 ¼ 3.1, P ¼ 0.03). Post hoc tests showed that only the highest naltrexone dose (20 lg) significantly reduced 4% sucrose consumption relative to saline control (P <0.05). Naltrexone did not significantly reduce sucrose consumption under other conditions (Fig. 5B D; all F <2,P >> 0.05). As for NAcc microinjections, the highest dose of naltrexone caused a trend toward larger naltrexoneinduced suppression of 4% sucrose consumption in the 4-0 group relative to the 4-20 group, but this did not reach significance (Fig. 5E; t 46 ¼ 1.8, P ¼ 0.08). Cannula placements were confined to the NAcc (Fig. 6A) or the VTA (Fig. 6B) for these two series of experiments. NAcc core and shell regions are known to subserve distinct functions (Kelley & Swanson, 1997; Floresco et al., 2006; Lecca et al., 2006), raising the possibility that naltrexone effects might differ as a function of infusion site within the NAcc. However, because few of our rats had cannulae confined to the NAcc shell (most implants were in the core, or included both core and shell in the two hemispheres), we were unable to draw conclusions about different naltrexone effects in the core and shell. Discussion Our data demonstrate that endogenous opioids carry information related to a learned difference in the relative value of an otherwise identical 4% sucrose solution. These data show clearly that endogenous opioid signaling is not related exclusively to the macronutrient content of food items. Endogenous opioid signaling can act to promote consumption of preferred food items, even when preference is entirely

5 1224 S. A. Taha et al. Fig. 4. Infusion of naltrexone into the NAcc has minor effects upon sucrose consumption.(a-d) Naltrexone infusion directly in the NAcc had few effects upon sucrose intake; the largest effect was seen for 4% sucrose consumption in the 4-0 group (A), where a trend toward decreased consumption following naltrexone was apparent (P ¼ 0.06).(E) Box plots (indicating 25th, median, and 75th percentile values) show the effects of the highest dose of naltrexone (40 lg) in reducing 4% sucrose consumption relative to saline control, comparing 4-0 and 4-20 groups. A trend toward a larger effect in the 4-0 group (P ¼ 0.07) was apparent. Fig. 5. Infusion of naltrexone into the ventral tegmental area has small effects upon sucrose consumption.(a-d) Naltrexone infusion directly in the ventral tegmental area caused a significant decrease in consumption only for 4% sucrose consumption in the 4-0 group; post hoc tests showed that the largest dose of naltrexone significantly reduced consumption relative to saline control (*P < 0.05). Naltrexone did not alter consumption in any other condition. (E) A trend toward a larger suppression of consumption in the 4-0 group relative to the 4-20 group (P ¼ 0.08) was apparent following administration of the highest dose of naltrexone. dissociated from the gustatory properties of the reinforcer; nonetheless opioid signaling may also play a special role in promoting consumption of food items high in particular macronutrients (such as fat; see below). Our results broaden the scope of behaviours known to be modulated by endogenous opioid signaling. The data that we report here are consistent with a number of experimental results suggesting that opioids play an important role in controlling food intake specifically through modulation of palatability. A consistent finding across many studies is that opioid antagonists selectively reduce intake of preferred food items. Thus, consumption of sweet saccharin or sucrose solutions is reduced relative to water intake following naloxone administration (Le Magnen et al., 1980); naloxone suppresses sweet chow consumption more potently than normal chow intake (Levine et al., 1995), and consumption of high-fat and or high-sugar foods is more potently reduced than consumption of normal chow following naloxone administration (Giraudo et al., 1993). Indeed, in a study where preferred and nonpreferred food items were offered concurrently, naltrexone decreased preferred food intake while slightly increasing nonpreferred food intake (Cooper & Turkish, 1989). Opioid antagonist-induced reductions in preferred food consumption occur in both sham-feeding and normal animals, demonstrating that opioid antagonists act on orosensory processing related to consumption itself rather than postingestional signaling such as gastric distention (Rockwood & Reid, 1982). Consistent with this data, administration of opioid agonists such as morphine increase preferred food intake. Morphine increases consumption of saccharin without affecting water intake (Calcagnetti & Reid, 1983), and more potently increases intake of sucrose or sweetened chow than standard chow (Evans & Vaccarino, 1990). Morphine effects can be dependent upon baseline preference, in that systemic morphine administration increases carbohydrate intake most in carbohydrate-preferring rats and fat intake most in fat-preferring rats (Gosnell et al., 1990). However, other studies have shown that systemically administered morphine causes macronutrient-specific effects, preferentially increasing fat intake (Welch et al., 1994); similar results have been reported for microinjection of opioid agonists directly into the NAcc (Zhang & Kelley, 2000). While our results do not rule out a specific role for opioid signaling in promoting macronutrient-specific consumption, the contrast paradigm that we have used in this study allows unambiguous dissociation of the sensory properties of a sucrose reinforcer from its relative value, and shows that endogenous opioid signaling can carry information related exclusively to the degree to which a sucrose solution is preferred.

6 Opioids encode relative value 1225 Fig. 6. Cannulae placements. (A) Reconstructed NAcc cannula positions ranged from anterioposterior +0.7 to +1.7 and mediolateral mm. Cannula positions spanned the shell and core subregions of the NAcc. (B) Reconstructed ventral tegmental area cannula positions ranged from anterioposterior )5.2 to )6.3 mm. For both A and B, anteroposterior position is relative to bregma. In our study, neither intra-nacc nor intra-vta naltrexone administration had large effects upon sucrose consumption, particularly relative to the robust suppression of consumption seen after systemic naltrexone injection. It may be that neither the NAcc nor the VTA is the critical site of opioid signaling mediating relative reward. While this possibility cannot be excluded, there is ample evidence that opioid signaling in each of these nuclei has potent effects upon food intake. Opioid agonists infused into the NAcc profoundly elevate food intake, with preferential effects upon palatable foods (Zhang & Kelley, 1997, 2002), and opioid agonists in the VTA also potentiate food intake (Noel & Wise, 1995; Echo et al., 2002; MacDonald et al., 2003). Still, opioid signaling in a number of other brain regions, including the nucleus of the solitary tract, parabrachial nucleus, lateral hypothalamus and amygdala (Stanley et al., 1988; Kotz et al., 1997; Giraudo et al., 1998; Wilson et al., 2003), also modulates food intake, and one of these brain regions may critically mediate opioid signaling underlying relative value. An alternative possibility is that opioid signaling underlying relative value occurs through a widely distributed network, with multiple brain regions acting in parallel to promote opioid-driven feeding. This hypothesis suggests that small effects seen with single-site microinjections (either NAcc or VTA) might additively suppress intake when both regions are injected simultaneously. Our study leaves open the question of the specific opioid receptor mediating effects on palatable food consumption, but there is ample evidence from previous studies that signaling through the MOR is most important in the regulation of palatable food intake. In both the NAcc and the VTA, the MOR-specific agonist DAMGO most potently elicits feeding (Zhang & Kelley, 1997; Echo et al., 2002), and MORspecific antagonists most potently reduce sucrose intake (Kelley et al., 1996; Ward et al., 2006). Interestingly, signaling through a distinct opioid receptor appears to exert effects on feeding that oppose those driven by MOR signaling: intra-nacc administration of a selective delta opioid receptor (DOR) antagonist increases sucrose consumption in sated animals (Kelley et al., 1996). This finding suggests a third possibility for the minimal effects on feeding we observed following intra-nacc naltrexone administration. Naltrexone is a nonselective opioid antagonist. Concurrent blockade of both endogenous mu and delta opioid signaling may produce competing effects upon food intake, with MOR antagonism acting to decrease intake and DOR antagonism acting to increase consumption, with the net result that sucrose intake is largely unchanged from baseline conditions. Enkephalin, which is abundantly expressed in a subset of NAcc medium spiny neurons, is a ligand at both the MOR and DOR, and is likely to provide the tonic signaling at both receptors in the NAcc that is inhibited by naltrexone administration. Further experiments with selective MOR and DOR antagonists will be useful in testing this hypothesis. Acknowledgements We thank P. M. Newton and L. H. Corbit for helpful discussions and comments on the manuscript and R. Van and V. N. Kharazia for histology assistance. This work was supported by a grant from the State of California for research on alcohol and substance abuse through the University of California, San Francisco, and by funds provided by the Wheeler Center for the Neurobiology of Addiction, the National Institute on Drug Abuse, and NARSAD. Abbreviations DAMGO, [D-Ala2, N-Me-Phe4-Gly5-ol]-enkephalin; DOR, delta opioid receptor; MOR, mu opioid receptor; NAcc, nucleus accumbens; VTA, ventral tegmental area.

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