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Am J Physiol Regul Integr Comp Physiol 284: R1260-R1268, 2003. First published January 9, 2003; doi:10.1152/ajpregu.00716.2002
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Vol. 284, Issue 5, R1260-R1268, May 2003

Restricted feeding with scheduled sucrose access results in an upregulation of the rat dopamine transporter

Nicholas T. Bello1,2, Kristi L. Sweigart3,4, Joan M. Lakoski2,3, Ralph Norgren1,2, and Andras Hajnal1,2

Departments of 1 Behavioral Science and of 3 Pharmacology, 2 Neuroscience Graduate Program, 4 Integrative Biosciences Graduate Program, The Pennsylvania State University, College of Medicine, Hershey, Pennsylvania 17033


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Recent studies suggest that the mesoaccumbens dopamine system undergoes neurochemical alterations as a result of restricted feeding conditions with access to sugars. This effect appears to be similar to the neuroadaptation resulting from drugs of abuse and may underlay some pathological feeding behaviors. To further investigate the cellular mechanisms of these alterations, the present study used quantitative autoradiography and in situ hybridization to assess dopamine membrane transporter (DAT) protein density and mRNA expression in restricted-fed and free-fed adult male rats. The restricted feeding regimen consisted of daily limited access to either a normally preferred sucrose solution (0.3 M) or a less preferred chow in a scheduled (i.e., contingent) fashion for 7 days. Restricted-fed rats with the contingent sucrose access lost less body weight, ate more total food, and drank more fluid than free-fed, contingent food, or noncontingent controls. In addition, these animals had selectively higher DAT binding in the nucleus accumbens and ventral tegmental area. This increase in protein binding also was accompanied by an increase in DAT mRNA levels in the ventral tegmental area. In contrast to the restricted-fed groups, no differential effect in DAT regulation was observed across free-fed groups. The observed alteration in behavior and DAT regulation suggest that neuroadaptation in the mesoaccumbens dopamine system develops in response to repeated feeding on palatable foods under dietary constraints. This supports the notion that similar cellular changes may be involved in restrictive eating disorders and bingeing.

nucleus accumbens; neuroadaptation; contextual cues; latent inhibition; bingeing; corticosterone; insulin


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

DOPAMINERGIC PROJECTIONS from the mesencephalic ventral tegmental area (VTA) that synapse on the nucleus accumbens (NAcc) are considered a critical substrate of central motivational systems (4, 28, 46, 47, 56). In addition to a role in integrating the motivational and motor components of appetitive behavior, the mesoaccumbens dopamine (DA) system is implicated in appetitive learning, conditioning to contextual cues (14, 23, 26), and in drug addiction and relapse (6, 11, 24, 29, 37, 53). Despite considerable interest in the function of this system in the neural control of feeding, little is known about mesoaccumbens neuroadaptation in the context of different dietary conditions and consumption patterns (4, 5, 33, 47).

Individuals with binge-eating disorder or bulimia nervosa self-impose periods of restricted intake, a feeding pattern that may lead to increased avidity for palatable foods (42). Indeed, rats with a history of cycles of food restriction and refeeding with palatable foods exhibit persistent binge eating with a specific preference for these palatable foods (19). Microdialysis experiments have shown that restricted-fed rats have lower basal extracellular DA levels in the NAcc, but an exaggerated release in response to feeding or amphetamine (43-45). In addition, food restriction attenuates the habituation of DA release in the NAcc that normally occurs with steady access to palatable food (2, 20, 55). These findings suggest that an increased preference for palatable foods under conditions of food restriction is accompanied by a change in the functioning of the mesoaccumbens DA system.

In recent experiments from the Hoebel laboratory (12, 13), rats on a restricted feeding regimen developed excessive glucose intake over a period of 30 days, increased D1 DA and µ1-opioid receptor binding in the NAcc, and had altered dopamine membrane transporter (DAT) binding in the midbrain. We observed that a shorter (7 days) limited access to sucrose in restricted-fed rats results in a decrease in DA D2/D3 receptor binding in the NAcc (3), suggesting that changes in the regulation of DA tone in the NAcc may be associated with intermittent ingestion of palatable foods. This plasticity is not limited to preferred meals. Recent findings from our laboratory revealed increased DA uptake during a regular chow meal if it was preceded with expected sucrose in restricted-fed rats (21). Repeated exposure to amphetamine also induces upregulation of DAT (51).

On this basis, we hypothesized that food restriction augments the effect of expected daily sucrose ingestion on the mesoaccumbens DA system, resulting in an upregulation of the DAT. Therefore, quantitative autoradiography and in situ hybridization were employed to assess DAT protein density and mRNA expression, respectively, in restricted-fed rats. These rats were subjected to a 7-day feeding regimen similar to our previous microdialysis and receptor studies (3, 21). The specificity of the stimuli (i.e., preferred sucrose solution vs. chow) and importance of time cues and the relevance of stimulus contingencies were addressed with additional controls.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Animals. Thirty-six male Sprague-Dawley rats (Charles River, Wilmington, MA), with an initial weight of 350-400 g, were individually housed and placed on a 12:12-h light-dark schedule (lights on 0700) throughout the experiment. During the handling period, which was 1 wk before the scheduled feeding protocol, all rats had ad libitum access to regular laboratory chow pellets (Rodent Diet-W 8604, Harlan Teklad, Madison, WI) and water. Throughout the entire experiment (including the acclimatization and scheduled feeding periods) all animals remained in their home cages except when daily body weights were measured between 0800 and 0830. All the procedures used in this experiment were approved by the Institutional Animal Care and Use Committee of the Pennsylvania State University College of Medicine and comply with the American Physiological Society's Guiding Principles for Research Involving Animals and Human Beings.

Experimental groups and feeding protocols. The experimental design included three independent variables, each with two components: 1) restricted or ad libitum food access, 2) a first meal of either a 0.3 M sucrose solution or lab chow pellets, and 3) a second chow meal that was contingent or noncontingent on the first. The sucrose solution (0.3 M; 10.26%) is highly preferred by rats, reliably releases DA in the NAcc (20, 21), and approximates the sugar content of soft drinks (e.g., Coke: 11.25%; Brisk Ice Tea: 9.6%).

Accordingly, after the acclimatization period, rats were divided into six groups as outlined in Table 1. The groups break down as follows. Half were food and water restricted, half were free fed. Two restricted-fed groups were presented daily for 7 days with either sucrose or chow at 1000-1020. Two hours later both groups had a 20-min access to chow [1220-1240; contingent sucrose food (CSF) and contingent food-food (CFF) groups, respectively; Table 1]. The third restricted-fed group served as a noncontingent control, receiving sucrose, food, or no stimulus at the two access sessions randomly across days [noncontingent sucrose food (NCSF) group; Table 1]. The two free-fed groups had sucrose either on schedule, at 1000 for 20 min, or randomly, 20 min anytime [contingent sucrose (CS) and noncontingent sucrose (NCS) groups, respectively; Table 1]. A final, naive group (Naive) had food and water ad libitum but no access to sucrose. All the restricted-fed groups (i.e., CSF, CFF, NCSF) had ad libitum access to chow and water for 2 h each afternoon (1400-1600), whereas the free-fed groups (i.e., CS, NCS) had ad libitum access to water during this time. The feeding protocols were introduced in a staggered fashion for pairs of animals in each group (i.e., 2 animals per subgroup/day for each condition on 3 consecutive days). This method made euthanasia of all the animals possible within the best matching time period across all conditions.

                              
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  		Table 1.   Overview of experimental groups

Behavioral measurements. In addition to daily body weight measurement, food, sucrose (where applicable), and water consumption were recorded for all access sessions for each animal throughout the 7-day feeding regimen. Daily cumulative food is expressed in units of grams consumed per 100 grams of body weight (g/100 g body wt).

Blood glucose and plasma hormone assays. On day 7, all restricted-fed and free-fed rats were presented with their respective first access stimuli and were decapitated between 1145 and 1220 (before the second stimulus presentation). Approximately 3 ml of trunk blood from each rat was collected into an EDTA (K3, 15%) vacutainer tube (Becton Dickinson, Franklin Lakes, NY). After 20 µl was removed for blood glucose assay (Elite Glucometer, Bayer, Elkhart, IN; Ref. 34), the remainder of the blood sample was gently agitated and maintained on ice until centrifugation at 3,000 rpm for 10 min. Plasma was then distributed into three microcentrifuge tubes (Fisher Scientific, Pittsburgh, PA) and stored at -80°C until the day of the assay. Standard radioimmunoassay kits were used to determine plasma corticosterone (Cort; sensitivity; 25 ng/ml; ICN Biomedical, Redding, CA) and insulin concentrations (sensitivity; 0.02 ng/ml; Linco Research, St. Charles, MO).

Histology. After rats were decapitated and blood was collected, brains were removed and immediately immersed in -40°C isopentane (2-methylbutane) and stored at -80°C. The brains were sectioned on a cryostat in the coronal plane at 20 µm and thaw-mounted on poly-lysine coated slides. The brain regions examined were from the dorsal and ventral striatum (1.7-1.1 mm from bregma, inclusive of the NAcc) and from the mesencephalon [-5.6 to -6.1 mm from bregma, inclusive of the medial ventral tegmental area (VTA) and the substantia nigra (SN); Ref. 40]. Sections from each brain region were mounted on multiple slides (4), with serial sections distributed across the slides so that each slide had every fourth section. For autoradiography and in situ hybridization, adjacent slides were selected and binding values from two adjacent sections (represented a distance of 80 µm) were analyzed and averaged for each structure. One slide, each with two sections rostral and two sections caudal to each target region, was stained for cresyl echt violet and served to determine anterior-posterior coordinates for selection of proper slides and slices for analytical comparisons. The remaining sectioned tissue was stored desiccated at -80°C until day of the assays.

Autoradiography. Using a protocol similar to Tella and colleagues (52), slide-mounted sections were taken out of the -80°C freezer and thawed for at least 30 min. Slides were then incubated for 90 min at 4°C in a buffer solution that contained protease inhibitor cocktail that consisted of 25 mg chymostatin, 54 µM leupeptin, 100 µM EGTA, 100 µM EDTA in 0.05 M dibasic/monobasic phosphate buffer with 20 mg BSA (Sigma, St. Louis, MO), and 32.5 pM of the radioligand 125I-labeled RTI-55 (2,200 Ci/mol; DuPont NEN, Boston, MA). One buffer bath contained the radioligand and 1 µM of paroxetine (GlaxoSmithKline, Pittsburgh, PA) to block RTI-55 binding to the serotonin transporters. To determine nonspecific binding, the other buffer bath contained the radioligand and both 1 µM of GBR-12935 (a dopamine-selective uptake inhibitor, 1-[2-(diphenylmethoxy)-ethyl]-4-(3-phenylpropyl)piperazine, Sigma) and 1 µM paroxetine. Immediately after the incubation, the labeled sections were washed three times for 20 min each in ice-cold 0.05 M PBS buffer. Slides were then dipped in double-distilled water and dried overnight at room temperature. Subsequently, the slides were placed in a cassette with 125I microscale standards (Amersham, Arlington Heights, IL) and apposed to Kodak biomax MR film (Eastman Kodak, Rochester, NY) for 30 h.

In situ hybridization. The cDNA rat DAT construct was a 3-kb fragment sequence (kindly provided by Dr. Susan Amara, Howard Hughes Medical Institute, Oregon Health Science University) that was amplified and inserted into pBluescript II SK- plasmid (Stratagene, La Jolla, CA). Amplification of the transcript, riboprobe synthesis, tissue processing, and hybridization techniques were conducted in a fashion similar to that described by Campbell and Hess (8a) with modifications as specified below.

In vitro transcription was performed at 37°C for 1 h in a 20-µl volume containing 40 nM Tris, pH 7.9, 6 mM MgCl2, 100 mM DTT, 0.5 µl RNA inhibitor (Promega Biotec, Madison, WI), 2.5 mM NTP (mix of ATP, UTP, GTP), [35S]CTP (1,250 Ci/mmol, DuPont NEN), 1 µg linearized template DNA, and 15-20 U of T3 RNA polymerase (Promega Biotec). After transcription, the DNA template was removed by digestion with RNase-free DNase (Promega Biotec) for 15 min at 37°C. The probe was then cut into smaller pieces by incubation for 45 min in a solution that contained 2 µl 10 mg/ml tRNA, 66 µl STE buffer, and 2N NaOH. The probes were then extracted with Tris-buffered phenol-chloroform (pH 4.3), and unincorporated nucleotides were removed by filtering with Boehringer quick spin columns (Roche, Indianapolis, IN).

The slide-mounted sections were pretreated with 4% formaldehyde solution for 10 min at room temperature followed by a 1-min rinse in 0.1 M PBS. The slides were then immersed in 0.1 M triethanolamine and, after 1 min, 0.25% acetic anhydride was added to the solution. The slides remained in this solution for a total of 10 min to neutralize the positive charges on the tissue and slides. The slides were then rinsed in 2× SSC (0.15 M NaCl, 0.015 M sodium citrate) for 1 min. After this, the sections were dehydrated in graded ethanol solutions followed by two 5-min incubations in chloroform. Air drying followed 1-min incubations in 100 and 95% ethanol.

Each slide was hybridized with 100 µl of buffer containing 6.5 ng of the DAT cRNA probe in 50% deionized formamide and 10 mM DTT. The slides were placed under coverslips and hybridized for 18 h at 55°C. After hybridization, the coverslips were removed and slides were washed for 10 min in 2× SSC at 55°C. The slides were then treated with 50 µg/ml pancreatic RNase A (Promega Biotec) in 0.5 M NaCl, 10 mM Tris, pH 8.0, and 5 mM EDTA for 10 min at 37°C. Immediately after this, slides were washed in graded salt solutions (2×, 1×, and 0.1×) at 55°C for 5 min each and dipped in a solution containing 60% ethanol and 0.33 M ammonium acetate. Air-dried sections were placed in a cassette with 14C-labeled microscale standards (Amersham, Arlington Heights, IL) and apposed to Kodak biomax MR film for 30 h.

Quantitative analysis. For both autoradiography and in situ hybridization, immediately after the exposure time elapsed, the films were developed using standard photographic procedure. Film images were captured and digitized with a Howtek scanner (MultiRad 850, Howtek, Hudson, NH). Tissue images were quantitated by a densitometry procedure using microscales for 125I and 14C, respectively, to generate a separate standard curve for each film and assay. Quantitative analysis was done with the personal computer-compatible analytical imaging station (Imaging Research, St. Catherines, ON, Canada) software. Binding and hybridization was assessed for a target region unilaterally in all tissues. Background and nonspecific or sense binding or both were subtracted from all assayed tissue, as appropriate.

Statistical analysis. Behavioral data was expressed as mean or normalized values ± SE and were analyzed by multivariate ANOVAs (MANOVAs) and two-way ANOVAs with repeated measures. Appropriate post hoc tests included Neuman-Keuls and least-significant difference tests with level of significance set at alpha  = 0.05. Results from the histological assays were analyzed by ANOVAs and Neuman-Keuls post hoc test for pair-wise comparisons where applicable (Statistica 5.0, Tulsa, OK).


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Body weight. The restricted feeding resulted in loss of body weight in all the subgroups subjected to the regimen [range 5-17%; F(5,29) = 9.02, P < 0.0001], with significant post hoc tests in both the restricted-fed and free-fed groups compared with the Naive (P < 0.001). Group-wise comparisons revealed that while NCSF rats lost 10% of their initial body weight by day 7 (body weights on day 1 and day 7: 355.5 ± 8.7 and 319.7 ± 3.6 g, respectively, P < 0.05), CSF rats lost only 5% (body weights on day 1 and day 7: 367.8 ± 11.7 and 348 ± 13.6 g, respectively, not significant). The difference between the two subgroups in body weight for the 7-day comparison was statistically significant [F(1,9) = 6.46, P = 0.032; Fig. 1A]. The restricted-fed rats that were controls for the contingency, but did not receive sucrose (CFF), lost body weight similarly to the NCSF rats [CFF vs. CSF: F(1,9) = 5.98, P < 0.05]. This indicates that neither sucrose presentation nor contingent feeding alone was sufficient to maintain body weight as high as the CSF rats. In free-fed controls (dotted lines in Fig. 1A), however, there was no difference in body weight with respect to whether sucrose was presented in time-locked fashion (i.e., CS) or randomly (i.e., NCS). These two findings together suggest that food restriction promotes a differential effect of contingent sucrose presentation on body weight regulation.


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  		Fig. 1.   Summary of the behavioral findings. A: body weights (means ± SE) as expressed in percent of the initial weights. B: cumulative normalized daily food intake over 6 days of the feeding regimen. Values represent total intake of chow including all feeding sessions of the day. C: absolute volume of daily sucrose intake, if applicable. D: absolute volume of total daily fluid intake (water plus sucrose, if applicable). Continuous lines indicate groups that were restricted fed, whereas dotted lines depict groups with ad libitum food access. Experimental groups and conditions are identical to those listed in Table 1. For statistics and further details, see RESULTS. Cont., contingency.

Total food intake. Because of the individual differences in body weight as well as the statistically significant treatment effect on body weight across subgroups, food intake data were normalized. Total food intake includes data from the 20-min morning feeding sessions (if applicable), and 2-h afternoon chow intake is depicted in Fig. 1B.

Different feeding conditions had an overall effect of reducing the daily total food intake compared with the naive controls [F(5,25) = 18.07, P < 0.0001] including an effect across free-fed controls on water deprivation. The MANOVAs with planned comparisons, however, showed that, whereas restriction conditions (i.e., contrast for CSF-NCSF vs. CS-NCS) have a differential effect on total food intake [F(1,15) = 32.74, P < 0.001], the contingency conditions per se (i.e., contrast for CSF-CS vs. NCSF-NCS) did not influence intake of food. A separate analysis within restricted-fed subgroups revealed that CSF rats consumed more food than NCSF and CFF rats, with the difference being statistically significant even after normalization of intake to the higher body weight of the CSF group (P < 0.05 for both comparisons; Fig. 4B). In contrast, food intake between ad libitum-fed groups CS and NCS did not differ statistically (dotted lines in Fig. 1B).

In summary, food restriction resulted in a differential increase in food intake in rats that had received food contingently paired with sucrose prefeeding compared with paired feeding with chow or noncontingent sucrose access.

Composition of daily total food intake. Additional analyses were aimed at possible differences in the composition of total daily food intake across subgroups. Specifically, the respective contribution of the first, second, and third meals (i.e., absolute intakes from 20-min presentations and from the 2-h afternoon access) were compared between all applicable groups (i.e., CSF and CFF groups; Fig. 2). The ANOVA showed an overall group effect between CSF and CFF groups (P < 0.05). Post hoc tests verified statistical differences in intakes during the second morning meal on days 2, 4, 5, and 6 (P < 0.03, P < 0.03, P < 0.04, P < 0.01, respectively; Fig. 2A). Similar comparisons for the 2-h normalized food intake in the afternoon (1400-1600) revealed higher intake of CSF rats on days 2, 3, and 4 (P < 0.03, P < 0.02, P < 0.03; Fig. 2B). This finding demonstrates that the tendency for CSF rats to consume more extends through the second meal to the afternoon feeding.


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  		Fig. 2.   Composition of the daily food intake throughout the 6-day restricted feeding period in contingent sucrose-food and contingent food-food rats. A: intake of chow during the second access period in the mornings (1000-1020). B: intake of chow during the afternoon 2-h period (1400-1600). Because of differentially changing body weight between groups (see Fig. 1A) data shown here are normalized to body weight and represents g food/100 g body wt. For statistical comparisons, see RESULTS.

An influence of a meal on the consecutive meal can be both caloric and metabolic. To address this point, the normalized calorie intake of the first meal (either sucrose or chow in group CSF and CFF, respectively) was compared between groups. The ANOVA showed no subgroup effect, however, indicating that caloric preload did not contribute to the differences in second meal size.

Sucrose intake. For statistical comparisons, sucrose intake was normalized to body weight. The ANOVA failed to demonstrate a group effect in sucrose intake across all groups. There was, however, a significant time effect found [F(3,57) = 53.25, P < 0.0001], reflecting an approximately threefold increase in sucrose intake over 7 days (2.2 ± 0.5 and 5.8 ± 0.7 ml/100 g body wt on day 1 and day 7, respectively). Absolute intake of sucrose for all groups is shown in Fig. 1C.

Total fluid intake. Analysis of total daily fluid intake was chosen as a parameter to compare repletion of body fluids across groups. Because there was no statistical difference in sucrose intake across subgroups, this comparison serves as a measure of the effect of treatment on the drinking behavior and fluid balance regulation. Comparison of the normalized daily total fluid intake revealed a strong group effect [F(4,24) = 8.85, P < 0.001; Fig. 1D]. Post hoc tests showed that CSF rats drank more than any other subgroup and that there were no differences in fluid intake across the CS, NCS, NCSF, and CFF groups.

Blood glucose, plasma corticosterone, and insulin levels. Blood glucose tests from blood samples that were taken on day 7 (2 h after the first access period and just before the second meal was scheduled) were all within the normal range and did not show group differences (CSF: 5.55 ± 0.13; NCSF: 5.67 ± 0.44; CFF: 5.82 ± 0.15; CS: 5.45 ± 0.3; NCS: 5.6 ± 0.19; Naive: 6.02 ± 0.2; values in mmol/l).

Plasma Cort levels varied across groups significantly [F(6,29) = 4.35, P < 0.005]. Cort was elevated in food-restricted rats (CSF, NCSF, CFF: 252.34 ± 44.27, 314.08 ± 75.38, 308.8 ± 163.11 ng/ml, respectively) relative to Naive (47.22 ± 4.02 ng/ml) and ad libitum-fed groups (CS, NCS: 116.67 ± 44.76 and 47.77 ± 4.36 ng/ml, respectively). A planned comparison for deprivation condition revealed a strong effect [F(1,30) = 12.13, P < 0.002]. Cort levels correlated negatively with body weight [r = -0.34, F(1,34) = 4.37, P < 0.05]. In summary, Cort was higher in restricted-fed rats, which, in turn, lost more weight, but was not directly affected by the rewarding value and the presentation of the meals.

Plasma insulin levels measured from trunk blood that was taken on day 7 were a function of food restriction [F(1,27) = 20.36, P < 0.001] and positively correlated with the body weight [r = 0.37, F(1,34) = 5.56, P < 0.03] but were not affected by the rewarding value of the meal or by the stimulus contingencies (CSF: 0.47 ± 0.13; NCSF: 0.38 ± 0.07; CFF: 0.29 ± 0.11; CS: 0.93 ± 0.47; NCS: 1.88 ± 0.28; Naive: 1.55 ± 0.51; values in ng/ml).

Dopamine transporter binding. The 125I-RTI-55 density in the VTA showed a significant group effect [F(5,27) = 2.91, P < 0.03]. In the VTA, binding in the CSF rats was greater than those in the NCSF, CFF, and Naive groups (P < 0.05, respectively; Fig. 3). Similarly, in the shell of the NAcc, an overall analysis of the binding of RTI-55 showed a significant group effect [F(5,27) = 2.45, P < 0.05; Fig. 2]. The binding density was the highest in the CSF group and the lowest in the NCSF group [F(1,8) = 8.37, P < 0.02]. Binding in the CSF group was higher than that of the CFF and Naive group [F(1,8) = 7.58, P < 0.05; F(1,8) = 6.92, P < 0.05, respectively; Fig. 2]. In contrast, there were no statistical differences in the NAcc shell across free-fed groups and between those groups and naive controls (hatched columns in Fig. 3). Taken together, these results indicate that stimulus contingency in combination with a preferred stimulus has a differential effect on 125I-RTI-55 binding in the shell of the NAcc, but only in the restricted-fed condition.


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  		Fig. 3.   125I-RTI-55 radioligand binding in 5 brain regions of different experimental groups. Brain regions examined were the ventral tegmental area (VTA), medial shell of the nucleus accumbens (NAcc Shell), the core of the nucleus accumbens (NAcc Core), the pars compacta of the substantia nigra (SN p.c.), and the dorsolateral striatum (DL-STR). Data reflect means ± SE and are expressed as percentage of naive controls (dotted line = 100%). Experimental groups are identical to those specified in Table 1. a Statistically significant differences between restricted-fed groups; b statistically significant differences compared with naive controls. For actual P values and for all other comparisons, see RESULTS.

In the core of the NAcc, the greatest 125I-RTI-55 binding density was observed again in the CSF animals (post hoc test for group effect: P < 0.02; Fig. 3), but in contrast to the shell and the VTA, there was no statistical difference between CSF and CFF rats. Furthermore, both contingently fed groups (i.e., CSF and CFF) maintained the same binding as the naive controls (Fig. 3). In addition, 125I-RTI-55 binding in the NAcc core of ad libitum-fed rats did not differ from naive controls irrespective of whether sucrose was presented randomly or in a contingent fashion (i.e., NCS vs. CS; hatched columns in Fig. 3). These findings together indicate that the effect on the core reflects contingencies, but only in the restricted-fed condition, and is not a function of the rewarding value of the stimulus in either feeding condition.

Finally, an analysis of DAT binding in two structures of the nigrostriatal DA system, the pars compacta of the SN, at the level of the VTA, and the dorsolateral striatum (DL-STR), at the levels of analyses for the NAcc, did not reveal a group effect across all conditions (Fig. 3). This indicates that the observed effects were specific to the mesoaccumbens DA system.

Dopamine transporter mRNA hybridization. In the VTA, the highest level of in situ hybridization of DAT mRNA was found in the CSF group compared with other restricted-fed groups (CSF vs. NCSF: P < 0.05; CSF vs. CFF: P < 0.02; Fig. 4). As with the protein binding assays, there were no statistical differences across free-fed groups and between those groups and naive controls (hatched columns in Fig. 4). This demonstrates that, in addition to an increased DAT protein availability at both the somato-dendritic and the terminal regions of the mesoaccumbens DA system, the effect of the different feeding conditions extended to transcriptional alterations.


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  		Fig. 4.   Hybridized dopamine transporter mRNA in the VTA of rats that were maintained on different feeding regimen for 7 days. Data reflect means ± SE and are expressed as percentage of naive controls. Experimental groups are identical to those specified in Table 1. Dotted line represents naive controls (100%). a Statistically significant differences between restricted-fed groups; b statistically significant differences compared with naive controls. For actual P values and for all other comparisons, see RESULTS.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Scheduled sucrose ingestion results in an upregulation of the DAT. The major finding of the present study was that a scheduled, short access to sucrose resulted in an upregulation of the mesoaccumbens DAT. This effect was apparent only in food-restricted rats and did not occur in free-fed rats that were given the same scheduled access to sucrose. Moreover, removing the time contingency with random sucrose presentations also eliminated the effect. Except for the CSF group, our results were consistent with the findings from the Figlewicz lab (39), in that we observed a reduction in the DAT levels in the VTA in food-restricted animals relative to free-fed controls. In short, our results suggest that access to sucrose in a fixed feeding pattern was necessary to reverse the suppressive effect of food restriction on DAT. Regular chow in place of sucrose (i.e., restricted-fed CFF group) failed to produce the same effect, indicating the specificity of sucrose.

Further analyses of these effects revealed anatomical differences. Among all structures investigated, only the VTA and NAcc shell were sensitive to all three experimental variables, i.e., food restriction, rewarding value, and contingency. The NAcc core reflected the effects of both food deprivation and stimulus contingency, but not of the rewarding value of the stimulus. This observation is in concert with the literature proposing differential roles for the compartments of the NAcc based on lesion studies (18, 25, 26, 38, 54) and microdialysis experiments (1, 8, 36, 41). Because we did not observe a differential binding effect of DAT across groups in the dorsolateral striatum, the involvement of striatal motor systems, as reflected in the regulation of DAT, appears not be influenced by the feeding conditions. Despite this, we did observe differential binding of D2-like receptors in the dorsolateral striatum under similar feeding conditions (3).

The upregulation to the DAT protein in the NAcc was accompanied by an increased expression of DAT mRNA in the VTA, suggesting an effect that is related to genomic processes. In contrast to studies from the Hoebel laboratory (12, 13) that used a 30-day feeding regimen, our observations were made on day 7 after 6 days of training with a restricted-feeding regimen. This shorter period seemed to be sufficient to induce changes to DAT availability. Notwithstanding, this time frame is consistent with the half-life of the DAT protein, which has been reported to be ~2 days (27).

Restricted sucrose ingestion increases feeding and body weight. The present data showed an orexigenic effect in CSF rats that also had higher DAT protein binding and mRNA expression. Overall, CSF rats ate and drank more than their restricted-fed contingency controls. Although this increased intake probably explains why the CSF rats maintained a higher body weight than the other restricted-fed groups, the underlying mechanism of this alteration in feeding behavior remains unknown.

Elevated plasma Cort has been demonstrated to increase accumbens DAT protein binding (48). It has also been reported that sucrose ingestion, similar to glucocorticoid replacement, normalizes the metabolic, behavioral, and neuromodulatory effects of adrenalectomy (31). Along with the present data indicating that CSF rats expressed an upregulated DAT and maintained a higher body weight, these findings suggest a role for adrenal steroids in metabolic and dopaminergic homeostasis. The Cort assays performed in this experiment, however, did not yield a difference between the restricted-fed groups that were presented with the sucrose-food or the food-food regimen. It is worth noting that this conclusion was based on a single measurement of Cort on day 7, which cannot reflect circadian and pulsatile variations (32).

Insulin is another putative candidate that could have differentially influenced the dopaminergic mechanisms between our feeding groups (7, 9, 12, 17). Moreover, an effect of insulin may also be aggravated by contextual cues (15). In the present experiment, however, plasma insulin and blood glucose levels measured on day 7 did not differ across groups. Future experiments are needed to address the contribution of the dynamic effects of insulin and blood glucose levels on the development of the altered DAT availability.

Food restriction alters effect of repeated sucrose, possibly by altering dopamine signaling. An alternative interpretation of the present findings is that food restriction altered the predictive cue function of sucrose. In other words, in the CSF rats, the predictive value of sucrose ingestion is likely to influence behavior at the subsequent meals. Indeed, we observed increased chow consumption after contingent sucrose in restricted-fed rats relative to noncontingent controls. In addition, no increased intake was apparent during the second meal in the CFF group. Although not directly assessed in the present design, this observation indicates a role for contextual cues rather than simple time cues. This possibility is further supported by the differences in DAT binding in the NAcc core of the CSF rats compared with CFF rats that were subject to the same time cues and deprivation conditions.

A preferred reward-predicting stimulus increases expectation-related activation of dopaminergic neurons more than a neutral stimulus (49). The salience of the stimulus also is critical for subsequent meals and may be affected by food restriction (2, 50). Indeed, food restriction prevents the habituation of the DA response that usually occurs with repeated access to palatable foods (2, 21, 55). A similar effect, a disruption of latent inhibition by repeatedly high accumbens DA, has been proposed in the sensitization effects of amphetamine to psychostimulants (35) and to appetitive stimuli (22). Although the potency of natural rewards to produce the full spectrum of behavioral and neurochemical effects of drugs of abuse (i.e., sensitization, tolerance, withdrawal) has been debated, shared features in the mechanisms of action of dopaminergic psychostimulants and natural rewards are obvious. In particular, although the primary effect of amphetamine is to release cytosolic DA, it also has been shown to induce upregulation of DAT mRNA (51). Similarly, both stimulation-evoked endogenous DA and locally applied DA have been demonstrated to increase DAT function (10, 57). Consequently, repeated DA stimulation together with faster reuptake may result in intermittent high DA pulses in the NAcc (30). Such a relative change in phasic vs. tonic DA transmission has been proposed for coding of the incentive value of a stimulus (16).

With this background and the present data, it appears that food-restricted rats consistently release high DA in the NAcc in response to contingent sucrose and that this is associated with a compensatory regulation of the DAT. This observation is supported by our previous results using the same paradigm that revealed a downregulation of the D2/D3 receptor, which could represent a decrease in presynaptic autoreceptors (3). Both these cellular events are adaptations to intermittent high DA release, but they may also increase synaptic signaling by increasing the signal-to-noise ratio (i.e., phasic vs. tonic DA). In this way, stimuli and related cues that originally have low incentive value may become more effective in eliciting consummatory behaviors. Although the observation in the present experiment that CSF rats consumed more water can be secondary to the increased chow intake, it may also indicate some generalization of the effect.

In conclusion, the present findings support the hypothesis that plasticity in the mesoaccumbens DA system occurs in response to repeated appetitive stimulation at least under certain dietary conditions. These conditions include restricted feeding and scheduled access to a preferred food. A similar neurochemical adaptation may contribute to adjustments to perturbed feeding conditions including daily exposure to snack foods and high-sugar beverages and also may underlie pathological processes, particularly in binge eaters and self-restricting bulimics.


    ACKNOWLEDGEMENTS

The authors thank J. Furfaro and E. Kauffman for excellent help with the autoradiography and in situ hybridization assays and K. Matyas and N. Horvath for histology.


    FOOTNOTES

This research was supported by National Institutes of Health Grants DC-04751, DC-00240, and AG-17477.

Present address of J. M. Lakoski: Department of Pharmacology, University of Pittsburgh, Pittsburgh, PA 15261.

Address for reprint requests and other correspondence: A. Hajnal, Dept. of Behavioral Science H181, College of Medicine, The Pennsylvania State Univ., Hershey, PA 17033 (E-mail: axh40{at}psu.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

First published January 9, 2003;10.1152/ajpregu.00716.2002

Received 21 November 2002; accepted in final form 6 January 2003.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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Am J Physiol Regul Integr Comp Physiol 284(5):R1260-R1268
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