We determined the effects of repeated intermittent access to sucrose on opioid and DA neurotransmission in mammalian brain. Longitudinal in vivo PET imaging of the μOR and DA D2/3 receptors revealed reduced receptor availability throughout the reward circuit, including the nucleus accumbens, prefrontal cortex, and the anterior cingulate cortex. The results clearly demonstrate that sucrose affects reward mechanisms in a manner similar to that of drugs of abuse.

The intake of sucrose as a palatable substance is known to release DA and induce dependency in rodents15, with sucrose shown to be even more pleasurable than cocaine in rodents in certain contexts. Thus, rodents work more intensely to obtain sucrose than cocaine, even in the absence of food deprivation5. However, the effects of sucrose are regulated both by the homeostatic system and by hedonic reward circuits16,17 that may mediate the distinction between nutritional and hedonic aspects of sucrose action18. We opted for a one-hour per day schedule in order to promote “binging”, as previous studies in rats had revealed a higher intake during the first hour of daily access in an intermittent schedule15,19. Behavioral studies of food intake often target food-restricted animals, but the design may not necessarily reflect the same neural mechanisms active in obesity. Pigs in the present study were not food restricted and were fed the usual amounts of their normal diet in addition to access to sucrose.

Opioid receptors (OR) are widely expressed in the brain, specifically in structures known to modulate eating and reward processes20. ORs have been shown to be important in the rewarding and relapsing effects of cocaine21,22,23,24. Alterations in binding have also been linked to the homeostatic responses to eating and the pleasure associated with palatable food25. In particular, “liking” of food is linked to the endogenous opioid system, especially the μOR9,10 in the shell of the nucleus accumbens and the ventral pallidum26. Infusions of a μOR agonist into distinct portions of the nucleus accumbens and ventral pallidum strongly enhance “liking” behaviors, including tongue protrusions and paw licking, following increased palatable intake of food27,28,29. Further evidence for opioid signaling in the processing of hedonic regulation comes from μOR antagonists that attenuate consumption of palatable chow in both ad libitum-fed and food restricted animals, but with a more limited effect on the intake of non-palatable standard pellets30,31. In humans, μOR antagonists decrease short-term food intake and reduce pleasantness of palatable foods32,33,34. Opioid signaling in the basolateral amygdala also contributes to food “wanting” through modulation of reward seeking and the incentive value of food35.

With [11C]carfentanil, we obtained images of tracer binding that is sensitive both to μOR levels and to the brain’s release of endogenous opioids36,37. We detected immediate loss of μOR availability in areas of the nucleus accumbens and anterior cingulate cortex, specific brain regions of the reward pathway, after initial consumption of sucrose by five minipigs, consistent with endogenous opioid release. Previous studies have shown that palatable food can lead to feelings of pleasure38 by stimulating opioid release. After 12 days of sucrose access, we observed decreased [11C]carfentanil binding, which has several possible explanations39 including endogenous opioid release and binding to μOR, μOR internalization as a result of increased opioid binding, and increased DA D2/3 receptor activation leading to heterologous desensitization of μOR40.

In support of the present findings, [11C]carfentanil studies of patients with bulimia41, obesity42,43,44, and binge-eating disorder45, show decreased receptor availability. However, these are chronic conditions whereas the minipigs only received sucrose for 12 days. In a study of acute feeding behavior in healthy men, feeding led to robust and widespread endogenous cerebral opioid release, both in the presence and absence of hedonia, suggesting that opioid release reflects metabolic and homeostatic, as well as hedonic, responses25. This study, together with another that imaged patients after a chocolate-flavored liquid meal44, is directly relevant to the acute study of five minipigs after the first sucrose exposure, but is different from the subchronic sucrose-exposure study over 12 days where the reduced receptor availability more likely reflects repeated overstimulation and concomitant downregulation of μOR.

The prefrontal cortex is important in decision-making and ascribing value to items and therefore the μOR in the prefrontal cortex may be accountable for the altered evaluation of food saliency, which can raise the addictive potential of food. We have found decreased binding in the prefrontal cortex, consistent with previous studies showing that high fat diet reduces levels of μOR mRNA in the prefrontal cortex46 and that infusion of a μOR agonist in the prefrontal cortex increases intake of sweet food47. Again, however, the issue arises whether the high fat diet is a more chronic condition that more likely mediates receptor down-regulation, compared to the shorter-term sucrose-feeding design, suggesting sustained release of endogenous opioids that displaces tracer carfentanil bound to μOR, even after 12 days of sucrose.

DA has been implicated in rewards both from drugs and behavior. Chronic cocaine use has been found to inhibit DA signaling48. DA D1 and D2/3 receptor levels are altered by nicotine in pig brain49, and in non-human primates with a history of cocaine abuse50, consistent with the downregulation of D2/3 receptors in the brains of human cocaine addicts51,52. As for drugs of abuse, sucrose has been shown to upregulate DA D1 receptors19 and increase DA release53, reinforcing the role of DA in “wanting” in relation to palatable food. Previous PET studies have demonstrated a decrease in striatal DA D2/3 receptor availability in morbid obesity vs average weight54,55, similar in magnitude to the reduction in drug-addicted patients56, and in animal with models of obesity57. In rodent studies, D2/3 receptor knockdown in the striatum promotes the development of compulsive food seeking in rats with access to palatable food57.

Our observations of decreased D2/3 receptor availability of the pig may indicate increased DA levels in response to the incentive salience associated with the sucrose intake since DA is released as part of the wanting of drugs of abuse and other pleasurable activities52,58,59,60. As the pigs were anesthetized during the imaging, and had not received sucrose in 24 hours, the decreased D2/3 BP ND more likely reflects a reduction in the number of receptors in response to prolonged increase of DA release at each of the 12 days of sucrose access. The reduction can raise brain reward thresholds, associated with down-regulation of striatal DA D2 receptors. This may explain the increased susceptibility to drugs of abuse seen in previous studies of rats overeating sucrose that led to cross-sensitization to cocaine, hyperactivity after low dose amphetamine, increased alcohol intake when abstaining from sucrose, and tolerance to the analgesic effects of opiates6.

A previous study of obesity in the Göttingen minipig identified decreased cerebral blood flow in the nucleus accumbens, ventral tegmental area (VTA) and prefrontal cortex, with single photon emission computed tomography (SPECT) of brain61. Consistent with these findings, we observed reduced DA D2/3 binding in the ventroforebrain region containing the nucleus accumbens and in the prefrontal cortex. Extracellular levels of DA are increased 3-fold in the nucleus accumbens after sucrose intake in freely-moving rats undergoing microdialysis62. In sucrose dependent animals, repeated sucrose intake can lead to release of DA from the shell of nucleus accumbens63. Animals fed a restricted diet with limited access to sucrose had lower DA D2 receptor binding in the nucleus accumbens shell and the dorsal striatum64. Restricted high fat and sucrose diets can lead to sustained downregulation of D1 and D2 receptor mRNA in the nucleus accumbens65. A microdialysis study of the effects of palatable food revealed increased DA release in the nucleus accumbens and prefrontal cortex when the food was still considered novel; once the rats were accustomed to the new food, the increased release was blunted in the nucleus accumbens, but not in the prefrontal cortex66. The differential susceptibility to habituation and conditioning of the activity in two regions may explain the larger increase observed in prefrontal cortex than in nucleus accumbens of minipigs exposed to the same palatable substance that lost novelty after twelve days. However, as we did not image minipigs with [11C]raclopride after the first sucrose administration, this explanation is speculative.

The prefrontal cortex modulates executive function, decision-making, and self-control67. Dysfunctional DA neurotransmission in the prefrontal cortex impairs modulation of reward processing, suggesting impaired executive function and decision-making skills in obese individuals68,69. Moreover, a human PET study correlated decreased frontal cortex metabolism with decreased striatal D2 binding in obesity70. Here, we find reduced D2/3 receptor availability in the prefrontal cortex including the orbitofrontal cortex of pigs exposed to the sucrose regimen.

Dopaminergic neurons of the VTA send projections to the hippocampus and amygdala, where they support habit-like behaviors71 and mediate the encoding and retrieval of conditioning to drug72,73 and food cues74,75. Human brain imaging has shown hippocampal activation in response to food craving and tasting76. Consistent with our findings of a reduced hippocampal and amygdalar D2/3 receptor availability in response to sucrose, human brain mapping with [18F]fallypride showed cocaine cue-induced DA release in amygdala and hippocampus77. In rodent brains, cocaine cue exposure triggered DA release in the amygdala78, and alterations of amygdala DA levels influenced cue-induced cocaine-seeking behavior79.

In a study of obese individuals, the association between D2/3 and μOR availabilities, known to exist in striatal regions of lean individuals, was disrupted in the ventral striatum80. We compared the values of BP ND of the two tracers to test if the data reproduced this effect. Unlike lean humans, the present brains of pigs had no correlation between the values of BP ND of the two tracers, at baseline or after the exposure to sucrose. We then tested whether the animals with the largest declines of tracer raclopride binding would also have the largest decreases of tracer carfentanil binding, but instead we found a negative correlation in the averaged extrastriatal regions, suggesting that animals with the greatest change of the binding potential of tracer raclopride had the lowest change of the binding potential of tracer carfentanil. The inverse relation between the changes suggests that the effects of sucrose intake on the availabilities of the respective receptors are regulated in opposite directions. It is known that excessive consumption of palatable food, or drugs, can be driven by wanting or liking, or both60,81. It is possible that the magnitude of wanting driven by dopamine negates the magnitude of liking driven by opioids, or vice versa. Recent evidence points to roles of GABA A receptors in the VTA and cholinergic terminals in striatum and possibly cortex that act as switches between dopamine-dependent and dopamine-independent mechanisms of opioid action82,83 that may explain the reciprocity of dopamine and opioid effects in porcine extrastriatal regions determined here.

A shortcoming of PET, also in comparatively large animals, is the limited spatial resolution of the tomography that affects the results from small brain regions involved in food-associated behaviors. However, despite these concerns, [11C]raclopride binding previously was recorded both in striatal and extrastriatal regions84,85,86,87. The use of [11C]raclopride to label the same type of receptors raises no concern about potential affinity differences that may affect the use of separate tracers for the same receptors in different regions. Recent studies included records of extrastriatal binding of [11C]raclopride. Alakurtti et al. found good reproducibility of measures of striatal raclopride binding in the striatum, with only good to moderate reproducibility in the cortex85. In a later study, Svensson et al. discussed several issues affecting the use of [11C]raclopride as a marker of extrastriatal D2/3 receptors in a study of healthy humans, including poor reproducibility in cortex and limited decline of extrastriatal binding in frontal cortex in response to a D2/3 blocking agent88. The test-retest comparisons revealed variabilities of 4–7% in striatum and 13–59% in cortical regions, but the time between examinations averaged 20 days, unlike the more informative 1–2 days of most studies. A number of factors in the lives of those subjects may have had time to influence the findings. Indeed, we show here that merely adding sucrose consumption to a morning routine for 12 days may have influenced binding measures obtained two weeks later. Other factors as common as playing video games, shopping, entering new romantic relationships and sexual activity, using drugs or changing diet and exercise may influence extrastriatal dopamine levels with potential for great variation of datasets. The current study in minipigs introduced a well-controlled set-up with the only variable being the absence or presence of sucrose in the diet. In this context, the data from seven animals had sufficiently low variability in relevant extrastriatal regions to identify a statistically significant reduction of binding in response to sucrose.

A limitation of the current study is the use of anaesthetics required to ensure immobility during in vivo imaging of animals. The effects of specific anaesthetics, and their interactions with drugs or other interventions, can confound the binding of radioligands89,90. Ketamine is an anti-glutamatergic drug with rapid antidepressant effects in sub-anaesthetic doses91,92,93, that do not reduce striatal [11C]raclopride binding in humans94. However, S-ketamine was found to reduce binding availability of dopamine D2/3 receptors in striatum of conscious non-human primates95. Isoflurane is a common anaesthetic in animal PET. In previous studies, we found striatal accumulation of [11C]SCH23390, a radioligand of the dopamine D1 receptors to be significantly higher in minipigs anesthetized with isoflurane rather than propofol, suggesting susceptibility of the dopaminergic neurotransmission to effects of anaesthesia96. In the current study, all minipigs were imaged at both timepoints under ketamine pre-medication and isoflurane anaesthesia, making the present comparisons valid.