We have previously demonstrated that exposure to high fat (HF) during early development alters the presynaptic regulation of mesolimbic dopamine (DA), and increases incentive motivation for HF food rewards. The goal of the present experiments was to examine the long-term consequences of early exposure to HF on anticipatory and consumatory nucleus accumbens (NAc) DA responses to HF food rewards. Mothers were maintained on a HF (30% fat) or control diet (CD; 5% fat) from gestation day 13 to postnatal day 22 when offspring from both diet groups were weaned and maintained on the CD until adulthood. In vivo NAc DA responses to food anticipation and consumption were measured in a Pavlovian conditioning paradigm using voltammetry in freely moving rats. HF-exposed offspring displayed reduced NAc DA responses to a tone previously paired with the delivery of HF food rewards. In an unconditioned protocol, consumatory NAc DA responses could be isolated, and were similar in HF and control offspring. These data demonstrate that exposure to HF through maternal diet during early development might program behavioral and functional responses associated with mesolimbic DA neurotransmission, thus leading to an increased HF feeding and obesity.
It is now recognized that pre- and postnatal maternal consumption of calorie-dense food increases the offspring’s susceptibility to the development of obesity,1 although the underlying mechanisms are still unclear. Mesolimbic dopamine (DA) neurotransmission, which mediates the rewarding properties of food and food cues, represents a possible candidate. DA function is altered in diet-induced obesity in both humans2, 3, 4 and animals,5, 6, 7, 8 and DA projections develop for a large part postnatally,9 making them susceptible to the ‘organizational effects’ of early diet. Along with other groups,10, 11 we previously demonstrated that exposure of the mother to HF during the last week of gestation and throughout lactation blunted locomotor and nucleus accumbens (NAc) DA responses to amphetamine, and reduced presynaptic expression of DA D2 receptors in adult offspring.12, 13 Behaviorally, these functional changes led to an increase in incentive motivation for HF food pellets, as measured with three reinforcement conditions in an operant conditioning paradigm.12
A large body of evidence indicates that increased NAc DA transmission is necessary to generate the behavioral responses elicited by food and signal anticipation rather than food consumption.14 In the present experiments, we tested the hypothesis that early exposure to HF through maternal milk programs NAc DA responses to food and food cues in the adult offspring. We used in vivo voltammetry in freely moving rats to monitor rapid fluctuations in extracellular NAc DA concentrations during the anticipation and consumption of HF food rewards in adult offspring exposed to HF during early development. We demonstrate that HF offspring display a reduction in their anticipatory, but not consumatory, DA responses to food, suggesting that the increased operant responding to fat pellets in these rats12 is a consequence of maternally programmed DA hypofunction.
Female Sprague–Dawley rats (Charles River, Quebec) were placed on powdered diets from Harlan Teklad (Madison, WI, USA). HF (30% fat, 24% carbohydrate, 15% protein, 4.54 kcal g−1, HF) or CD (5% fat, 60% carbohydrate, 15% protein, 3.45 kcal g−1) diet was given from gestation day 13 to postnatal day (PND) 22. Litters were culled to 10 pups, weaned at PND 22 and fed the CD until testing. Animals were housed under controlled conditions of light (12:12 h light:dark cycle), temperature and humidity. The procedures were approved by the Animal Care Committee at McGill University.
Recording electrodes consisted of 30 mm Nafion-coated carbon fibers placed in a glass capillary, as previously described.14 Prior to implantation, electrodes were calibrated in vitro. Specificity was tested with ascorbic acid and measured by increasing the concentrations of DA. Only electrodes with a minimum specificity for DA of 1000:1 and a highly linear response to DA (r>0.9997) were implanted. Electrodes aimed at the shell of the NAc (lateral: −1.6 mm, anterior-posterior: +1.6 mm, dorsal-ventral: −7.6 mm from bregma) and Ag/AgCl reference electrodes aimed at the contralateral parietal cortex were implanted under isoflurane anesthesia. Pin connectors were soldered to both the electrodes and inserted into a plastic strip connector anchored to the skull. Electrode placements were confirmed postmortem using the atlas of Paxinos and Watson.15
Electrochemical recordings were performed as previously described,14 using a computer-controlled high-speed chronoamperometric instrument (FAST16, Quanteon, Lexington, KY, USA). An oxidation potential of +0.55 V (with respect to the reference electrode) was applied to the electrode for 100 ms at a rate of 5 Hz. The amplitude of the oxidation current was digitized and integrated over the last 80 ms of the pulse. Currents were averaged and converted into μM concentrations using the in vitro calibrations. The concentration of DA (μM) at the onset of each tone was used to calculate the mean change in signal (μM) for 10 s prior and 60 s following this timepoint.
Animals were tested in adulthood (>PND 90) in the dark phase of the light:dark cycle and 3–4 days post surgery. Animals underwent 4 days of conditioning with 15 trials per day. Each trial consisted of a 30-s 90-dB tone. At 30 s, a ‘click’ arose from the food dispenser and a 45-mg HF pellet (dustless precision pellets, 45 mg 35% fat, Bio-Serv, Frenchtown, NJ, USA) was delivered. DA oxidation currents were measured throughout the 60 trials. In the unpaired condition, trial conditions were similar, but the cues did not predict the delivery of the pellet. A variable inter-trial paradigm was used in both conditions.
Reduced anticipatory DA responses to food cues in HF vs control offspring
Mean change in NAc DA signal (μM) on days 2, 3 and 4 of conditioning is illustrated in Figures 1a and b (controls and HF, respectively). In both diet groups, repeated pairing of the compound cue (tone and ‘click’) with the delivery of a food pellet led to an anticipatory DA response. In both diet groups, modest but significant increases in DA were observed during the 30-s tone. The end of the tone, coupled with the ‘click’ of the food dispenser, induced a large rise in DA concentrations with a peak that occurred 2–3 s after the ‘click’. The dropping of the pellet into the food hopper occurred ∼2 s after the click, which coincides with the feeding onset. Thus, we can attribute the large DA increase observed to an anticipatory rather than a consumatory response. Our results agree with the previous research demonstrating that NAc DA transmission is activated primarily by conditioned cues that reliably predict a positive outcome (that is, receiving or earning food), and that this activation ceases once the expected food is presented and consumed.14 More importantly, our results demonstrate that NAc DA responses to the tone (Figure 1c) and the ‘click’ (Figure 1d) were decreased in HF vs CD offspring on day 4 of conditioning, although no significant differences were observed in earlier days. This effect cannot be attributed to learning deficits, as rats in both diet groups immediately consumed the pellet upon delivery on day 4 of testing.
The diminished DA response to the cued presentation and subsequent consumption of HF food rewards in adult offspring exposed to HF during early development is remarkable, as these offspring were only exposed to HF during the preweaning period. This suggests that perinatal and postnatal programming of NAc DA function modified the anticipatory response to food cues, thus potentially influencing the overall food consumption in the paired condition. NAc DA hyporesponsiveness in HF offspring extends to modalities other than food cues, as we previously found reduced locomotor and NAc DA responses to amphetamine.12, 13
Our results parallel the earlier reports in humans2, 3, 4 and rodents5, 6, 7, 8 showing that diet-induced obesity is associated with reduced DA function. Whether this hyporesponsive DA function results from the development of obesity or is a factor predisposing individuals to the development of obesity remains unclear. Our results suggest that maternal diet and the resulting perinatal nutritional environment can program DA function, and that NAc DA hypofunction can occur prior to the development of obesity, as our HF rats were not obese when tested. Furthermore, electrically evoked DA release was also found to be reduced in mesocorticolimbic terminal regions of obesity-prone rats.16 Together, these data support the hypo-sensitivity to the reward hypothesis of obesity, which postulates that in individuals with blunted DA function, the excessive consumption of palatable foods serves to reach a threshold of reward contributed by mesolimbic DA activation.17 The dissociation between anticipatory DA responses and operant behavior towards the same fat rewards12 is interesting, and suggests that in HF offspring, the role of glutamate in modulating operant responses to food in association with DA might be enhanced.18
Similar consumatory DA responses in the unpaired condition between control and HF offspring
On day 4 of testing in the unpaired condition, when the compound cue did not predict the arrival of the food pellet, DA responses to the cue were close to zero in both control and HF offspring (Figure 2a). We found, however, that a DA peak could be consistently observed when animals consumed the fat-enriched pellets. This peak was isolated and 15 data points prior to and following this peak were used for analysis (Figure 2b). No diet group differences were observed in a ‘pure’ consumatory response. Although not directly compared in the present analysis, peak consumatory responses in the unpaired condition were significantly smaller than anticipatory peak responses in the paired condition (0.4 vs 0.2 μM), especially in the control offspring, again demonstrating that NAc DA transmission is activated primarily by conditioned cues that reliably predict a positive outcome.
Our results indicate that exposure to HF during a critical period of development programs mesolimbic DA function in that adult offspring originating from mothers exposed to HF during the last week of gestation and throughout lactation display reduced anticipatory NAc DA responses to food cues, but no differences in consumatory DA responses. These changes in DA neurotransmission occurred prior to the development of obesity in these animals, suggesting that DA hypofunction programmed in early life might have a causal role in behavioral adaptations geared towards obesity. Our data provide strong evidence for the long-term nutritional ‘programming’ of the rewarding properties of fat-enriched rewards and associated mesolimbic DA function. This might lead to alterations in ingestive behavior that favor the development of obesity.
This research was supported by a grant from CIHR (no. 84299) to C-DW and AG. LN is a recipient of a CIHR Canada Graduate Scholarship.