Moderate High Fat Diet Increases Sucrose Self-Administration In Young Rats (2013)

Dianne P. Figlewicz,^1,2 Jennifer L. Jay,² Molly A. Acheson, Irwin J. Magrisso,⁴ Constance H. West,¹ Aryana Zavosh,² Stephen C. Benoit,³ and Jon F. Davis³

We have previously reported that a moderately high fat diet increases motivation for sucrose in adult rats. In this study, we tested the motivational, neurochemical, and metabolic effects of the high fat diet in male rats transitioning through puberty, during 5-8 weeks of age. We observed that the high fat diet increased motivated responding for sucrose, which was independent of either metabolic changes or changes in catecholamine neurotransmitter metabolites in the nucleus accumbens. However, AGRP mRNA levels in the hypothalamus were significantly elevated. We demonstrated that increased activation of AGRP neurons is associated with motivated behavior, and that exogenous (third cerebroventricular) AGRP administration resulted in significantly increased motivation for sucrose. These observations suggest that increased expression and activity of AGRP in the medial hypothalamus may underlie the increased responding for sucrose caused by the high fat diet intervention. Finally, we compared motivation for sucrose in pubertal vs. adult rats and observed increased motivation for sucrose in the pubertal rats, which is consistent with previous reports that young animals and humans have an increased preference for sweet taste, compared with adults. Together, our studies suggest that background diet plays a strong modulatory role in motivation for sweet taste in adolescent animals.

Subjects

Subjects were male Albino rats from Simonsen (Gilroy, CA). Rats were maintained on chow (Laboratory Rodent Diet 5001, LabDiet) or moderate high fat diet (31.8%; Research Diets Inc) ad libitum. The diets are matched for overall carbohydrate content (58% kcal vs. 51%kcal for low fat vs. high fat, respectively). The low fat chow has 6.23 gm% free sugars and the high fat diet has 29 gm% sucrose. They were maintained on a 12:12 h light-dark cycle with lights on at 6 AM. Unless otherwise indicated, rats were brought in at 3 weeks of age, immediately post-weaning, and were housed for acclimation until 5 weeks of age. At this age, diet and/or behavioral training and testing were begun. Specific protocols are described in detail below, and summarized in Table 1. Because male rats go through puberty at the 6^th-7^th week of age, the timing of studies was designed to study rats as they traverse this developmental stage. All procedures performed on the rats followed the NIH guidelines for animal care, and were approved by the Animal Care and Use Sub-Committee of the Research and Development Committee at the VA Puget Sound Health Care System.

  

Table 1

Experimental Protocols

Sucrose self-administration

General Protocol. Procedures were based upon our published methodology (Figlewicz et al., 2008; Figlewicz et al., 2006). All training and testing procedures were carried out between 0700 and 1200 hr. The experiment included 2-3 phases: autoshaping and fixed ratio (FR) training; surgery and recovery in specified cohorts (see Table 1); and progressive ratios (PR) training using the PR algorithm of Richardson and Roberts (Richardson & Roberts, 1996). The PR algorithm requires 1, 2, 4, 6, 9, 12, 16, 20, 28, 36, 48, 63, 83, 110, 145, 191, 251, 331, 437, 575, 759, 999, 999(etc) lever presses for succeeding reward deliveries within a session, and is a rigorous test for motivation and reward (27). Rats were trained to self-administer 5% sucrose (0.5 ml reward) delivered into a liquid drop receptacle. The operant boxes, controlled by a Med Associates (Georgia, VT) system, had two levers, but only one lever (an active, retractable lever) activated the infusion pump. Presses on the other lever (an inactive, stationary lever) were also recorded. The sucrose solution was delivered into a liquid drop receptacle for oral consumption (Med Associates). Initial training was conducted during one-h sessions for 10 days under a continuous reinforcement schedule (FR1: each lever press was reinforced), with a maximum possible of 50 sucrose rewards delivered per session. Each session began with the insertion of the active lever and the illumination of a white houselight that remained on for the entire session. A 5-s tone (2900 Hz, 20 dB above background)+light (7.5 W white light above the active lever) discrete compound cue accompanied each reward delivery, followed by a 20-sec time out after each sucrose delivery. PR training was carried out for a maximum possible 3 h/day for ten days. Daily sessions ended after 30 min of no active lever press responding, at which point the house light was turned off and the active lever retracted.

Effect of AGRP on sucrose self-administration

As our results showed an increase of AGRP mRNA expression in pubertal rats fed the high fat diet, we wanted to confirm that AGRP could increase sucrose self-administration. 5-wk old chow-fed rats were taken through FR training, then received cannulas into the third cerebral ventricle (ICV). Following a week of recovery, confirmation of placement with an angiotensin II drinking response test (see Figlewicz et al., [2006]), and one session of FR re-training, rats were started on the PR self-administration paradigm. After PR Day 1, rats were assigned to one of two groups such that mean PR Day 1 performance did not differ between the two groups (artificial CSF vehicle, aCSF; or AGRP, 2 μl of 0.01 nmol). They received injections of aCSF (n=8) or AGRP (n=7) on PR days 2, 5, and 8. Total daily food intake was quantitated during the PR training time.

Effect of age on sucrose self-administration

We compared self-administration behavior between the pubertal rats and young adults, fed chow or the 31.8% fat diet. Rats had two weeks of acclimation to the VAPSHCS vivarium (3—5wk or 8—10 wk). They then received the diet during the entire test/training period (4 wk). Thus, as in the initial experiment, pubertal rats were studied at 5-8 wk of age. Young adults were studied at 10-13 wk of age.

Body composition determination

Body composition was measured using quantitative magnetic resonance spectroscopy (QMR [Nixon et al., 2010]) to determine the body water content of individual rats, from which relative body fat is calculated. The animals were placed in cylindrical holders un-anesthetized, and then the holders are inserted into the QMR machine for a 2 minute scan, which performs triplicate measurements. Data are saved to an integrated computer (EchoMRI, Echo Medical Systems, Houston, TX) for immediate calculation of whole-body water, fat, and lean mass.

Intravenous glucose tolerance testing (IVGTT)

Conscious IVGTTs were carried out in rats with chronically implanted IV cannulas, that were fasted overnight prior to study, utilizing methodology based upon Frangioudakis, Gyte, Loxham, & Poucher (2008). Bilateral intravenous cannulas were implanted two weeks prior to study, as per our established methodology (Figlewicz et al., 2009). Baseline samples were drawn at t-10 min (0.5 ml for determination of insulin and glucose, at all time points) and t0 min. Rats received an infusion of 1 gm glucose/2ml/kg over 15-20 seconds followed by 0.5 ml flush of saline. Blood samples were taken at 5, 15, 30, 60, 90, and 120 min. Owing to plugging of a catheter during the procedure (hence, inability to obtain blood samples), final ‘n’s for the baseline/IVGTT data presented are 7-8 for chow-fed rats and 8 for rats fed 31.8% fat diet (Table 3). Plasma insulin was determined using Linco rat insulin RIA kits (#RI-13K and SRI-13K, Linco) and plasma glucose was determined on a YSI Glucose Analyzer). Area under the curve (AUC) for the response from baseline was calculated at 5 min and 120 min. The HOMA index was calculated as fasting (glucose [mM] × insulinm[U/L]) / 22.5 and was calculated using terminal fasting samples measured for insulin and glucose.

  

Table 3

Metabolic Parameters¹

Fasting metabolic parameters

Rats from Experiment 1 were fasted overnight prior to euthanasia, a few days after completion of the IVGTT. Rats were deeply anesthetized with isoflurane inhalation and exsanguinated. Brains were rapidly removed and frozen in liquid nitrogen for measurement of hypothalamic peptide mRNA and nucleus accumbens catecholamines. Terminal plasma or serum were used for measurement of fasting insulin, glucose, leptin, and triglycerides. For triglycerides, Point Scientific Triglyceride GPO Kit #T7531-400 (Fisher #23-666-418) and standards KIT #7531-STD (Fisher #23-666-422) were utilized, and 3 μl of serum was assayed in duplicate. Plasma leptin was measured with Millipore Linco RIA Kit# RL 83K.

Catecholamine HPLC Methods [Davis et al., 2008]

Rats were euthanized with isoflurane anesthesia, and brains were rapidly removed, frozen, and stored at −80 °C. Bilateral micro-punches of the nucleus accumbens (NAcc) were isolated from each animal. Although substantial care was taken to minimize contamination by neighboring brain regions, due to the nature and size of each micro-punch our method did not allow us to distinguish subregions (i.e. NAcc core vs. shell) within the NAcc. For high-performance liquid chromatography (HPLC) analysis, an antioxidant solution (0.4 N perchlorate, 1.343 mM ethylenediaminetetraacetic acid (EDTA) and 0.526 mM sodium metabisulfite was added to the samples followed by homogenization using an ultrasonic tissue homogenizer (Biologics; Gainesville, VA). A small portion of the tissue homogenate was dissolved in 2% sodium dodecyl sulfate (SDS) (w/v) for protein determination (Pierce BCA Protein Reagent Kit; Rockford, IL). The remaining suspension was spun at 14,000 g for 20 min in a refrigerated centrifuge. The supernatant was reserved for HPLC.

Samples were separated on a Microsorb MV C-18 column (5 Am, 4.6_250 mm, Varian; Walnut Creek, CA) and simultaneously examined for DA, 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA), both of which are markers of dopamine degradation, 5-HT and 5-HIAA. Compounds were detected using a 12-channel coulometric array detector (CoulArray 5200, ESA; Chelmsford, MA) attached to a Waters 2695 Solvent Delivery System (Waters; Milford, MA) under the following conditions: flow rate of 1 ml/min; detection potentials of 50, 175, 350, 400 and 525 mV, and; scrubbing potential of 650 mV. The mobile phase consisted of a 10% methanol solution in distilled H₂O containing 21 g/l (0.1 M) citric acid, 10.65g/l (0.075 M) Na2HPO4, 176 mg/l (0.8 M) heptanesulfonic acid and 36 mg/l (0.097 mM) EDTA at a pH of 4.1. Unknown samples were quantified against a 6-point standard curve with a minimum R² of 0.97. Quality control samples were interspersed with each run to ensure HPLC calibration.

Orexigenic peptides mRNA qPCR

We measured expression of hypothalamic peptides that stimulate feeding and have been implicated in motivation and reward behaviors (Figlewicz & Sipols, 2010): neuropeptide Y (NPY[ Hahn, Breininger, Baskin, & Schwartz, 1998; Jewett et al., 1995; Kelley, Nannini, Bratt, & Hodge, 2001]); agouti-related peptide (AGRP [Aponte, Atasoy, & Sternson, 2011; Barnes, Argyropoulos, & Bray, 2010; Broberger et al., 1998; Hagan et al., 2000; Hahn, Breininger, Baskin, & Schwartz, 1998; Kaushik et al., 2011; Krashes et al., 2011; Rossi et al., 1998; Tracy et al., 2008]); and orexin (Cason et al., 2010; Choi, Davis, Fitzgerald, & Benoit, 2010). Rats were euthanized with isoflurane anesthesia, and brains were rapidly removed, frozen and stored at −80° C until processing. The medial and lateral hypothalamus were microdissected as one block using an AHP-1200CPV freezing plane (Thermoelectric Cooling America, Chicago, Il) which maintained a constant temperature of 12° C throughout the dissection process. Total RNA from microdissected tissue was isolated by Trizol reagent (Invitrogen, Carlsbad, CA) and purified using the RNeasy Mini Kit (Qiagen, Valencia, CA) according to manufacturer’s instructions. The total RNA was treated to remove any potential genomic DNA contamination using RNase free DNase (Promega, Madison, WI), and was quantitated using a NanoVue spectrophotometer (GE Healthcare, Cambridge, UK). RNA quality was confirmed by standard agarose gel electrophoresis. Complementary DNA (cDNA) was then retrotranscribed (RT) from 1-2 μg of total RNA by a mixture of random hexamers and oligo DT priming using the iScript cDNA Synthesis Kit (Bio-Rad Laboratories, Inc., Hercules, CA). Non-retrotranscribed (no RT) reactions were also prepared from each sample to control for potential genomic DNA contamination. The cDNA and no-RT controls were diluted, and 5-10 ng of template cDNA from each sample was used to measure mRNA expression of selected genes by real time quantitative PCR utilizing the MyIQ Real-Time PCR Detection System (Bio-Rad, Hercules, CA).Triplicate measurements for each sample were run on standard iCycler 96 well plates, along with no template controls (NTC) to detect potential cross contamination, in 20 μl reaction volumes consisting of 10 μl 2× iQ Sybr Green Supermix (Bio-Rad, Hercules, CA), 2 μl of 0.2-0.5 μM each primer, 3 μl DEPC water, and 5 μl of template. All qPCR reactions included a melt curve analysis to ensure specificity of signal. Relative expression for each gene of interest was calculated by extrapolation to a standard curve individually run on each plate and derived from serial dilutions of a pooled sample of reference cDNA, and normalized to relative expression of reference genes (acidic ribosomal phosphoprotein 36B4 for gene expression in hypothalamic tissue, and mitochondrial ribosomal protein L32 for expression in the nucleus accumbens). The following primer sequences (IDT, San Diego, CA) were used to amplify rat prepro-orexin, NPY, and AGRP: Prepro-orexin, Forward: 5′-TTCCTTCTACAAAGGTTCCCT-3′, 5′-GCAACAGTTCGTAGAGACGGCAG-3′; NPY: Forward, 5- TACTCCGCTCTGCGACACTACATC-3′; Reverse: 5′-CACATGGAAGGGTCTTCAAGCC-3′; AGRP, Forward: 5′-GCAGAAGGCAGAAGCTTTGGC-3′; Reverse: 5′-CCCAAGCAGGACTCGTGCAG-3′.

cFos Immunocytochemistry (ICC) and Quantitation

Fluorescence ICC was used to identify Fos-positive and AGRP-positive neuronal cell bodies in the medial hypothalamus, according to our established methodology (Figlewicz et al., 2011). On the final day (PR Day 10), rats were placed in their self-administration chambers as usual, for 90 min. Immediately following that last 90 min session, rats were deeply anesthetized with isoflurane inhalation and perfused with 0.9%NaCl followed with cold 4% paraformaldehyde solution. The timing for anesthetic and euthanasia was based upon the known timecourse of peak expression of cFos protein at 90–120 min-post event. Thus cFos expression would reflect the activation of the CNS at the onset of the behavioral task, rather than being the result of the animals experiencing the task. Brains were removed and post-fixed in paraformaldehyde several days, then subsequently placed in 20% sucrose-PBS, then 30% sucrose-PBS solution. Brains were sectioned on a cryostat (Leica CM 3050S cryostat) for immunohistochemistry. We used our established methodology to quantitate immunoreactive cFos protein in brain sections (Figlewicz et al., 2011). Slide-mounted 12 μm whole-brain coronal sections were washed three times in phosphate buffered saline (PBS, OXOID, Hampshire, England). Sections were washed for 20 min with 100% ethanol/DI water (50%, v/v) followed by a PBS wash, then blocked for 1 hour at room temperature in PBS containing 5% normal goat or donkey serum. Sections were then washed multiple times in PBS and incubated overnight at 4°C in primary antibody solutions made up in PBS. Sections were washed three times in PBS and then incubated in the dark at room temperature in secondary antibody solution made up in PBS for 1 hour. Sections were subsequently washed again in PBS, and mounted and cover-slipped in Vectashield hard set mounting medium (Vector; Burlingame, CA) mounting medium. Digital images of sections were acquired using a Nikon Eclipse E-800 fluorescence microscope connected to a Qimaging Retiga digital capture camera using NIS Elements (Nikon) software.

Based upon PCR studies demonstrating increased AGRP mRNA levels, we focused upon medial hypothalamic regions, particularly the ventromedial nucleus and the arcuate nucleus (ARC)). Atlas-matched 12 μm sections were evaluated for cFos expression and quantitation in matched sections and regions, based upon the atlas of Paxinos and Watson [2005]. For quantitation (at 40× magnification), atlas-matched regions were selected. NIS Elements software (Nikon) was utilized to capture an image of the desired area. An area was delineated for counting and threshold for positive cell counts was established. The identical area and background (threshold) were utilized for sections from the respective experimental groups, and software counting of positive cells (quantitation) was carried out in the same session for all experimental groups, to prevent between-session changes in background setting. For statistical analysis, counts were taken from an individual rat only if corresponding or complete sections through each area were available; data for a specific area were not taken from a rat if there was incomplete bilateral representation for that area.

In addition to cFos quantitation, quantitative double-label immunohistochemistry for cFos and AGRP was conducted. Because we did not wish to disturb the animals’ behavioral performance, they were not pre-treated with colchicine to optimize visualization of AGRP. Therefore visualization of AGRP-positive neurons might be underestimated. The dual staining procedure for AGRP was comparable to the assay of cFos-immunoreactivity on its own, except that sections were blocked for one hour at room temperature in PBS-5% donkey serum. Then, a mixture of fos-Ab and AGRP primary antibodies was used for overnight incubation at 4°C; likewise both secondary antibodies were in the same solution and incubated for one hour in the dark at room temperature. Initial optimization assays were carried out to determine an appropriate dilution of the primary antibodies. Primary antibodies used were rabbit anti-cFos (1:500) (sc-52) and goat anti-AGRP (1:100) (18634) (Santa Cruz Biotechnology, Inc., Santa Cruz, CA). Secondary antibodies used were Cy3-conjugated donkey anti-rabbit (Jackson Immunoresearch; West Grove, PA), and Alexa fluor 488 donkey anti-goat IgG (Molecular Probes, Eugene, OR); all secondary antibodies were diluted at 1:500.

Statistical analyses

Group data are presented as means ± standard error of the mean (SEM) in the text, Tables, and Figures. Significance is defined as p ≤ 0.05. Statistical comparisons are made between experimental groups, as presented under “Results” using unpaired Student’s ‘t’test (e.g., diet, age, or treatment comparison). ‘Normalization’ of data is defined as it is used.

Effect of moderate high fat diet on peri-pubertal motivation for sucrose

Rats fed the 31.8% fat diet during wks 5-8, while in self-administration sessions, had a significantly elevated motivation for sucrose, compared with chow-fed rats. As shown in Figure 1a, there was no difference in performance during the initial FR training (averaged FRDays 1-10 active lever presses, 38±5 vs. 39±2 for chow vs. 31.8% fat diet, respectively). However, when the rats were switched to the more stringent PR task, there was a significant increase of number of active lever presses, and number of sucrose rewards taken, but not in overall session length (Figure 1b). There was no effect of the chronic diet treatment on the number of inactive lever presses. When rats were fed the high fat diet during wks 5-8 but subsequently returned to a chow diet taken through FR and PR training during weeks 9-12, there was a trend but no significant difference in active lever presses. Thus, there appears to be no behavioral carry-over effect of a moderately high fat diet consumed during the peri-pubertal timeframe. PR parameter data for these cohorts are summarized in Table 2. In order to begin to elucidate contributing mechanism(s) to the diet-induced increase in sucrose motivation, we carried out a number of metabolic and CNS measurements.

  

Figure 1

PR motivated responding for sucrose rewards is increased in peripubertal rats fed a 31.8% fat diet (n=8). 1a. Across the FR sessions, there was no effect of diet, but the diet effect is manifest when rats are switched to the PR paradigm. 1b. Data are …

  

Table 2

Effect of Peri-Pubertal High Fat Diet on Progressive Ratios Performance for Sucrose

Effect of moderate high fat diet on metabolic parameters

Immediately after the conclusion of behavioral testing, body fat composition was determined on rats that had the diet intervention and behavioral paradigm during wks 5-8. Rats then received chronic intravenous cannulas for (conscious) IV glucose tolerance tests (IVGTTs). Subsequently, terminal fasting plasma and serum were obtained for additional metabolic measures. As shown in Table 3, there were no differences in body composition, body weight, fasting insulin or glucose measures, insulin sensitivity (HOMA calculation) or responses to the IVGTT, between the chow-fed and high fat diet-fed rats. Terminal fasting leptin and triglyceride measurements did not differ between the two groups. Thus, although the diet treatment had a significant effect on motivation for sucrose, it reflects a behavioral response in high fat-fed rats that are pre-obese.

Effect of moderate high fat diet on CNS homeostatic and reward neurochemistry

In addition to terminal metabolic measurements, brains from the cohort that had both diet intervention and behavioral training during weeks 5-8 were measured for nucleus accumbens amine profiles (n=4 per diet group) or mRNA levels of hypothalamic orexigenic peptides. As shown in Table 4, there was no significant effect of the high fat diet on dopamine, norepinephrine, or serotonin metabolites in the nucleus accumbens, a central site of reward and motivational activity (Ikemoto & Panksepp, 1996; Kelley & Berridge, 2002) in which each of these neurotransmitter systems plays a key regulatory role. Within hypothalamic extracts, mRNA levels of the orexigenic peptides, NPY, AGRP, and orexin were measured. A strong but non-significant trend for increased AGRP in the fat-fed rats was observed in this cohort (n=8 for either diet); we therefore repeated the diet/behavioral training paradigm in an additional cohort and measured NPY, AGRP, and orexin mRNA in the hypothalamus. In the combined cohorts, we observed a significant (p<0.05) increase in AGRP mRNA in rats fed the high fat diet vs. chow controls (Figure 2), but no significant change in NPY or orexin expression. To evaluate possible connections between AGRP expression and self-administration behavior, we measured cFos and AGRP immunopositive neurons in the mediobasal hypothalamus. Groups of rats were fed the chow or 31.8% fat diet; some were taken through the self-administration protocol (weeks 5-8) and others were handled as behavioral controls. Figure 3a shows an example of co-localization of cFos and AGRP in an arcuate nucleus neuron. As summarized in Table 5, activation of AGRP neurons (co-expression of cFos-ICC and AGRP-ICC within the same cells) was associated with self-administration activity. This is demonstrated in Figure 3b, where the number of activated (cFos-positive) neurons is shown as the neuronal cell count, or as percent of total AGRP-positive neurons: there is significant activation of AGRP neurons in the rats self-administering sucrose, vs. the handling controls, in the combined diet groups. A within-diet treatment comparison for the number of activated AGRP neurons in the self-administration group vs. handling controls displayed a trend that did not reach statistical significance (chow, p=.078; 31.8% fat diet, p=.073). Importantly, not only do these data link AGRP neuronal activation with self-administration behavior, but because of the timing for the cFos measurement (90 minutes after the rats were placed in their self-administration chambers), cFos expression reflects activity of AGRP neurons in anticipation of, or at the onset of, self-administration activity. There was a non-significant trend for increased total AGRP-positive neurons in the self-administration group (vs. handling controls, p=0.16). In those rats, where lever-pressing was matched between the diet groups, the number of AGRP-positive neurons was also matched. There was no effect of the diet treatment alone on number of AGRP-positive neurons in the behavioral control rats.

  

Figure 2

Effect of 31.8% fat diet on medial hypothalamic peptide mRNA expression. Data are normalized for high fat-fed rats (n=17) vs. that of chow controls (n=16). AGRP mRNA is significantly elevated (p<0.05).

  

Figure 3

Activation of AGRP neurons at the onset of sucrose self-administration. 3a. Co- localization of cFos and AGRP in arcuate nucleus neuron, 60x magnification. 3b. Number of activated (cFos-immunopositive) AGRP-immunopositive neurons in the mediobasal hypothalamus …

  

Table 4

Nucleus Accumbens Amine Metabolites

  

Table 5

Agrp Neuron Activation: Diet and Behavioral Treatment

Effect of AGRP administration on sucrose motivation

Our interpretation of this finding is that AGRP expression in the pubertal rats is a key mechanism underlying the enhanced sucrose self-administration of the high fat diet-fed rats. To confirm for the efficacy of AGRP to increase motivation for sucrose, AGRP was administered via the third ventricle to chow-fed peri-pubertal rats during the PR portion of the behavioral paradigm. This dose regimen of AGRP was sub-threshold for stimulation of chow intake across the two weeks of PR paradigm, but resulted in significantly increased sucrose self-administration, as shown in Figure 4. (Note that each sucrose reward has a caloric content of 0.1 kcal, therefore the sucrose self-administration activity contributes negligible calories to total daily intake.) Table 6 shows self-administration parameter data across the 9-day PR paradigm, with AGRP or aCSF injected ICV on Days 2, 5, and 8. In AGRP-treated rats, the number of active lever presses was significantly increased overall across PR Days 2-10 (p=0.03), and on non-injection days (p=0.048) with a trend towards an increase on the (averaged) injection days. Additionally, Stop Time (which reflects the total time spent engaged in the self-administration task) was significantly increased on non-injection days (p=0.02) with trends towards an increase overall, and on injection days. The number of sucrose rewards was increased overall across PR Days 2-10 (p=0.03). There was no effect of AGRP treatment on inactive lever pressing, compared with aCSF-treated controls, or between injection- and non-injection days. The results support an interpretation of a sustained effect of AGRP to increase sucrose self-administration: rats pressed more on the rewarding lever, received more sucrose rewards, and spent more time engaged with the task.

  

Figure 4

Third ventricular (ICV) AGRP (0.01 nmol) stimulates sucrose self-administration in the PR paradigm, but has no effect upon daily food intake across the study period (PR Days 2—10, with injections on Days 2, 5, and 8). AGRP (n=9) data are expressed …

  

Table 6

Effect of ICV AGRP vs. aCSF on Progressive Ratios Performance for Sucrose

Effect of life stage on preference and motivation for sucrose

In the final experiment, we evaluated whether motivation for sucrose differs between pubertal and adult rats. Initially, 5-and 10-wk old rats were given a sucrose preference test with choices of solutions ranging from 0 to 20% sucrose, prior to beginning self-administration testing and training. As shown in Figure 5a, and consistent with findings reported in the literature, the pre-pubertal rats appeared to prefer a sweeter solution than the young adult rats: most pre-pubertal rats had a peak intake of 20% sucrose solution, whereas the adult rats showed a peak intake of 15% sucrose. Subsequently, both age groups were split between rat chow and high fat diet during the self-administration training and testing. There was a small but statistically significant increase in the number of active lever presses by the peri-pubertal vs. adult rats (45±3 vs. 37±2, p=0.05) averaged across the FR sessions, with no difference in number of sucrose rewards or number of presses on the inactive lever. As shown in Figure 5b, there was a highly significant overall effect of age, across the PR sessions, with significantly increased active lever pressing for the pubertal (n=15) vs. young adult (n=14) rats (2-way ANOVA, PRDay × age; effect of age, p=0.017, no independent effect of PRDay, no significant interaction). Tthere was a trend for a greater effect of age in the high fat diet-fed condition but this did not achieve statistical significance (p=.13). Table 7 lists PR behavioral parameters: in addition to increased active lever presses, peri-pubertal rats received significantly more sucrose rewards, and showed a trend towards increased Stop Time. Additionally, the peri-pubertal rats had a small but significant increase in presses on the inactive (i.e., non-rewarding) lever, although for both peri-pubertal and adult rats, the number of inactive lever presses was approximately 10% of the number of active lever presses. These results suggest that peri-pubertal rats prefer and will more avidly seek out sweet-tasting foods, and the effect may be amplified with a background of high fat diet.

  

Figure 5

Juvenile rats have increased motivation for sucrose rewards compared to adult rats. 5a. Sucrose preference tests for juvenile (peri-pubertal, n=15) and young adult (n=14) rats. Rats had 30 min to drink from the range of concentrations (0-20% sucrose). …

  

Table 7

Effect of age on on Progressive Ratios Performance^a for Sucrose

This research was supported by NIH grant DK40963. Dianne Figlewicz Lattemann is a Senior Research Career Scientist, Biomedical Laboratory Research Program, Department of Veterans Affairs Puget Sound Health Care System, Seattle, Washington. Stephen Benoit was supported by NIH DK066223 and Ethicon Endosurgery Inc. The authors thank Dr. Tami Wolden-Hanson for support with body composition measurements; Dr. William Banks and Lucy Dillman for support with the triglyceride measurements; and Amalie Alver, and Samantha Thomas-Nadler for assistance with the behavioral studies.

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