INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

FEEDING IS A COMPLEX behavior regulated by numerous brain regions and neuromodulators, including opioids. Opioid agonists stimulate feeding (12), and alterations in food intake or diet composition affect gene expression or peptide levels of endogenous opioids (42). Recent evidence has led to the hypothesis that opioid involvement in feeding may lie in the rewarding aspects of feeding, rather than in those related to energy requirements (20). The rewarding aspects of feeding are regulated not only by various neuromodulators, but by diet composition as well. Operant and free-feeding studies indicate that dietary sucrose is particularly rewarding (see Ref. 3 for review).

Because sucrose consumption is considered to be rewarding and because the reward pathway in the brain involves endogenous opioids, it has been hypothesized that consumption of sucrose induces the release of endogenous opioids. Several studies support this contention, largely on the basis of observations that various behavioral effects of the opioid antagonist naloxone are enhanced after sucrose consumption. Because these effects of naloxone are observed in the absence of exogenously administered opioids, this increased potency of naloxone presumably reflects antagonism of elevated levels of endogenous opioids. For instance, chronic consumption of a high-sucrose diet increases the ability of naloxone to inhibit operant responding for a food reward (35). Similarly, rats allowed to drink a sucrose solution show increased sensitivity to the appetite-suppressive effects of naloxone compared with rats with no access to sucrose (14, 19).

Other studies indicate that acute sucrose ingestion induces an immediate release of endogenous opioids, which affects behaviors other than feeding. Rat pups receiving an intraoral infusion of a 7.5% sucrose solution showed decreased pain sensitivity compared with those drinking water (33). Similar analgesic effects of sucrose consumption have been reported in human infants (2, 5). Also, termination of sucrose feeding to morphine-dependent rats increases the severity of naloxone-precipitated withdrawal, suggesting that a regimen of sucrose feeding enhances opioid tone (36). Furthermore, acute or chronic sucrose consumption has been shown to enhance (4, 15) or decrease (10) the analgesic properties of morphine. These effects on morphine-induced analgesia apparently are specific to dietary sucrose or carbohydrates, inasmuch as alterations in dietary proteins or vitamins and minerals have no effect on morphine-induced analgesia (13).

The present study tested the hypothesis that sucrose consumption increases endogenous opioid release by examining c-Fos immunoreactivity (c-Fos-IR) after naloxone administration to rats that had been subjected to prolonged exposure to a 10% sucrose solution. This methodology can be used to unmask brain regions under tonic opioid inhibition on the basis of the following rationale: Inasmuch as the effects of opioid agonists on cellular physiology are inhibitory, resulting in decreased activity of adenylate cyclase, increased K⁺ conductance, and decreased Ca²⁺ conductance (8, 27), interruption of these signals by naloxone would be expected to increase cellular activity and, hence, increase c-Fos-IR. An example of this hypothesis is as follows: A single opioid receptor-expressing cell with unoccupied opioid receptors that is treated with naloxone should show no alterations in cellular physiology. That same cell, when exposed to an opioid peptide, would be inhibited as described above. However, if this cell (when inhibited by an opioid peptide) is treated with naloxone, a release from inhibition would occur and may result in subsequent expression of immediate early genes such as c-fos. Thus elevated opioid tone due to sucrose ingestion may be seen as increased c-Fos-IR after naloxone injection (Fig. 1).

View larger version (20K): [in this window] [in a new window]   Fig. 1.   Schematic representation of detection of endogenous opioid tone. In brain regions under tonic opioid inhibition (A), interaction of an opioid receptor with endogenous opioid peptide results in activation of Gi/Go proteins with resultant inhibitory effects. When opioid antagonist naloxone displaces endogenous opioid peptide (B), these inhibitory processes may be reversed, resulting in disinhibition and, ultimately, c-fos expression.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Animals. Twenty-eight male Sprague-Dawley rats (Harlan, Madison, WI), weighing 225-240 g at the start of the experiment, were housed in individual cages in a temperature-controlled vivarium on a 12:12-h light-dark cycle (lights on at 0700). Rats were divided into two groups: the first group received normal chow and tap water ad libitum; the second group received normal chow and 10% sucrose solution ad libitum, with no access to water for 3 wk. Between 0900 and 1300 of the final experimental day, rats were given a subcutaneous injection of isotonic saline or 10 mg/kg naloxone hydrochloride (RBI, Natick, MA). Rats were not allowed to eat or drink for 1 h after injection. One hour after injection, rats were deeply anesthetized with pentobarbital sodium (Nembutal) and transcardially perfused as described previously (31). This design yields four treatments (n = 7/group): those drinking tap water and receiving a saline injection (water-saline), those drinking tap water and receiving a naloxone injection (water-naloxone), those drinking sucrose and receiving a saline injection (sucrose-saline), and those drinking sucrose and receiving a naloxone injection (sucrose-naloxone). One rat in the sucrose-naloxone group was excluded from the study because of illness before the completion of the experiment.

c-Fos immunohistochemistry. Frozen sections were cut on a cryostat at a thickness of 40 µm and were immediately placed in cryoprotectant (41) and subsequently stored at 20°C until staining. Sections were stained for c-Fos-IR, as described previously (31). Briefly, sections were rinsed in PBS, then incubated with anti-c-Fos antiserum (Oncogene Science, Cambridge, MA) at a dilution of 1:50,000 for 48 h at 4°C. After further rinses in PBS, sections were incubated in biotinylated anti-rabbit IgG at a dilution of 1:200 (Vector Laboratories, Burlingame, CA) for 1 h, rinsed, and then incubated for 1 h in Vectastain Elite ABC reagent (Vector Laboratories), rinsed, and then reacted with nickel sulfate-diaminobenzidine. To control for possible variations in the chromogen reaction, all sections in a given neural region were processed at the same time.

Data collection and statistical analysis. c-Fos-IR was examined in specific forebrain, midbrain, and hindbrain areas that are involved in food intake regulation or opioid dependence/withdrawal. In the forebrain, c-Fos-IR was examined in the central (ACe), basomedial (BMA), and basolateral nuclei of the amygdala, the laterodorsal (BNSTLD) and medial divisions of the bed nucleus of the stria terminalis, the caudate putamen (CPu), the ventral, intermediate, and dorsal divisions of the lateral septum, the core (NAccC) and shell (NAccSh) of the nucleus accumbens, the medial and lateral (latARC) divisions of the arcuate nucleus, and the dorsomedial (DMH), ventromedial (VMH), lateral, and paraventricular nuclei of the hypothalamus. In the midbrain and hindbrain, c-Fos-IR was examined in the ventral, intermediate, and lateral periaqueductal gray (latPAG), the locus coeruleus (LC), the lateral parabrachial nucleus (lPBN), the intermediate and rostral divisions of the nucleus of the solitary tract, and the intermediate and rostral divisions of the ventrolateral medulla.

	RESULTS

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Effects of sucrose consumption on c-Fos-IR. As shown in Table 1, there was a main effect of drink in a number of brain regions studied, typically described by an increase in c-Fos-IR in rats drinking sucrose compared with rats drinking water. The majority of these regions are located in limbic forebrain regions but also extended to the hindbrain. A significant main effect of drink was seen in NAccC [F_(1,21) = 10.124, P = 0.0045], NAccSh [F_(1,21) = 8.637, P = 0.0078], CPu [F_(1,21) = 6.503, P = 0.0186], ACe [F_(1,23) = 7.539, P = 0.0115; Fig. 2A], BNSTLD [F_(1,23) = 5.00, P = 0.0353; Fig. 2B], and lPBN [F_(1,18) = 8.240, P = 0.0102]. In all these regions, c-Fos-IR was significantly increased in rats drinking sucrose. There was also a significant main effect of drink in BMA [F_(1,23) = 9.073, P = 0.0062]; however, in this region, sucrose consumption decreased c-Fos-IR.

View larger version (28K): [in this window] [in a new window]   Fig. 2.   Naloxone-induced c-Fos immunoreactivity (IR) in central nucleus of amygdala (A) and laterodorsal division of bed nucleus of stria terminalis (B) is potentiated by sucrose consumption. Values are means ± SE. * Significantly different from water-naloxone group.  Significantly different from water-saline group.

Effects of naloxone injection on c-Fos-IR. As shown in Table 1, there was a main effect of drug in a number of brain regions, but this effect was restricted to discrete nuclei within the hypothalamus and amygdala. In all these regions, the main effect was due to increased c-Fos-IR in rats receiving naloxone injections compared with rats receiving saline injections. In the hypothalamus, a significant main effect of drug was seen in latARC [F_(1,23) = 8.027, P = 0.0094], VMH [F_(1,23) = 5.300, P = 0.0307], and DMH [F_(1,23) = 6.005, P = 0.0223]. In the amygdala, there was a significant main effect of drug in ACe [F_(1,23) = 169.00, P < 0.0001; Fig. 3] and BNSTLD [F_(1,23) = 25.935, P < 0.0001; Fig. 4].

View larger version (97K): [in this window] [in a new window]   Fig. 3.   Photomicrographs showing effect of sucrose consumption on naloxone-induced c-Fos-IR in central nucleus of amygdala. c-Fos-IR is shown in rats drinking water and given a saline injection (A) or a naloxone injection (B) and in rats drinking sucrose and given a saline injection (C) or a naloxone injection (D). Consumption of sucrose significantly potentiated naloxone-induced increase in c-Fos-IR but had no effect on c-Fos-IR in saline-treated animals. Scale bar, 200 µm. L, lateral; D, dorsal; M, medial; V, ventral.

View larger version (105K): [in this window] [in a new window]   Fig. 4.   Photomicrographs showing effect of sucrose consumption on naloxone-induced c-Fos-IR in basolateral division of bed nucleus of stria terminalis. c-Fos-IR is shown in rats drinking water and given a saline injection (A) or a naloxone injection (B) and in rats drinking sucrose and given a saline injection (C) or a naloxone injection (D). Scale bar, 200 µm.

Sucrose consumption alters naloxone-induced c-Fos-IR in ACe and latPAG. Of all the brain regions examined in this study, only two showed a significant drink × drug interaction (Table 1). The most pronounced effect was seen in ACe [F_(1,23) = 13.394, P = 0.0013]. c-Fos-IR in this area was significantly elevated in the water-naloxone group compared with the water-saline group [t₍₁₂₎ = 7.136, P <0.0001], indicating a basal level of endogenous opioid release. c-Fos-IR was also significantly higher in the sucrose-naloxone group than in the sucrose-saline group [t₍₁₁₎ = 10.937, P < 0.001]. To determine whether sucrose consumption significantly enhanced this opioid release, c-Fos-IR in the sucrose-naloxone group was compared with that in the water-naloxone group. Indeed, c-Fos-IR was significantly higher in the sucrose-naloxone group than in the water-naloxone group [t₍₁₁₎ = 3.505, P = 0.0049]. Along with these quantitative differences, qualitative differences in the pattern of c-Fos-IR were also present. In ACe, c-Fos-IR in the water-naloxone group was generally localized to the lateral division, whereas c-Fos-IR in the sucrose-naloxone group was spread throughout the lateral, medial, and capsular divisions (Fig. 3).

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Feeding influences on endogenous opioid tone in the amygdala. The data presented here provide indirect evidence in support of the hypothesis that sucrose consumption increases endogenous opioid tone in ACe. We propose that this elevated opioid tone, in turn, plays a significant role in the palatable and rewarding characteristics of sucrose.

View larger version (47K): [in this window] [in a new window]   Fig. 5.   Schematic representation of brain regions that showed a main effect of drink (A), a main effect of drug (B), and a drink × drug interaction (C). Areas showing a main effect of drink typically are associated with taste and reward pathways; areas showing a main effect of drug are associated with food intake regulation and energy balance. Central nucleus of amygdala (arrowheads) is only region showing main effects of both drink and drug and a drink × drug interaction.

Convergence of sucrose- and naloxone-mediated signals on ACe. Although there were significant main effects of drink and drug in ACe and BNSTLD, there was a striking divergence of areas showing a main effect of drink and those showing a main effect of drug (Fig. 5). This separation of responsiveness correlates with previous data on the roles of these regions in behavioral and physiological roles in taste, reward, and feeding. A number of areas that showed a main effect of drink have established roles in gustatory processing. After gustatory primary afferents enter the nucleus of the solitary tract, the taste pathway bifurcates at lPBN: one path follows a classic sensory thalamocortical route; the other projects to limbic regions, including ACe and BNSTLD (26). With the exception of BMA, the increased c-Fos-IR seen in these areas due to sucrose consumption was unaffected by naloxone injection, indicating involvement of non-opioid systems. If the increased c-Fos-IR in lPBN seen in the present study reflects activity of the taste pathway, then it is not surprising that such activity is unaffected by naloxone. Although naloxone has been shown to decrease intake of a 10% sucrose solution (29), it is unlikely that this effect is due to alterations in taste perception, inasmuch as naloxone does not alter the ability of rats to distinguish small amounts of sucrose from water in an operant discrimination task (28).

Sucrose-induced alteration of opioid tone. If, as we contend here, sucrose consumption increases endogenous opioid tone, then prolonged sucrose consumption may lead to a situation that is in some way analogous to opioid dependence. Administration of naloxone to rats drinking sucrose resulted in increased c-Fos-IR in some, but not all, of the same regions known to be activated after naloxone administration to morphine-dependent animals (1, 7, 9, 22, 32, 37). Although the pattern of c-Fos-IR seen in the sucrose-naloxone group resembles that seen in animals undergoing withdrawal, no changes in c-Fos-IR were seen in several prominent nuclei implicated in opioid dependence. Notable examples are LC and the septal nuclei. An absence of c-Fos-IR in LC in the sucrose-naloxone group is not entirely surprising, since the animals did not show withdrawal signs such as wet dog shakes, diarrhea, ptosis, weight loss, and jumping (data not shown). It appears that autonomic regions mediate these physiological responses of withdrawal, whereas limbic regions affect motivated behaviors. It has been shown that naloxone administration into LC induces severe physical withdrawal with relatively few changes in drug reward. However, naloxone injection into NAccC elicits almost no withdrawal behaviors but dramatically reduces drug reward (16). Furthermore, microinjection of methylnaloxonium (a congener of naloxone) into discrete brain sites induces some, but not necessarily all, withdrawal behaviors, depending on the site of injection. For instance, injection into LC induces severe withdrawal, whereas injection into ACe induces relatively mild withdrawal (23).

Perspectives