Enhanced sensitivity to drugs of abuse and palatable foods following maternal overnutrition

Epidemiological studies have shown an association between maternal overnutrition and increased risk of the progeny for the development of obesity as well as psychiatric disorders. Animal studies have shown results regarding maternal high-fat diet (HFD) and a greater risk of the offspring to develop obesity. However, it still remains unknown whether maternal HFD can program the central reward system in such a way that it will imprint long-term changes that will predispose the offspring to addictive-like behaviors that may lead to obesity. We exposed female dams to either laboratory chow or HFD for a period of 9 weeks: 3 weeks before conception, during gestation and lactation. Offspring born to either control or HFD-exposed dams were examined in behavioral, neurochemical, neuroanatomical, metabolic and positron emission tomography (PET) scan tests. Our results demonstrate that HFD offspring compared with controls consume more alcohol, exhibit increased sensitivity to amphetamine and show greater conditioned place preference to cocaine. In addition, maternal HFD leads to increased preference to sucrose as well as to HFD while leaving the general feeding behavior intact. The hedonic behavioral alterations are accompanied by reduction of striatal dopamine and by increased dopamine 2 receptors in the same brain region as evaluated by post-mortem neurochemical, immunohistochemical as well as PET analyses. Taken together, our data suggest that maternal overnutrition predisposes the offspring to develop hedonic-like behaviors to both drugs of abuse as well as palatable foods and that these types of behaviors may share common neuronal underlying mechanisms that can lead to obesity.

with maternal nutritional exposure the data of the two sexes were combined for the final analyses.
All testing was conducted during the dark phase of the light-dark cycle.

Conditioned place preference (CPP)
The apparatus used to carry out the conditioned place preference (CPP) consisted of two large chambers containing explicitly different visual and tactile cues (wall color and floor material) that were connected by a small shuttle chamber (neutral environment). A digital camera mounted above the CPP apparatus captured images at a rate of 5 Hz and transmitted them to a personal computer running the Ethovision tracking system (Noldus Technology), which calculated a mobility score defined as the distance travelled per bin in successive 5 min bins. Additionally, the total time spent in the compartment was indicated in seconds. All mice underwent a pre-conditioning test (Pre Test) allowing them to freely explore the entire apparatus for one 15 min session. The measure for the Pre Test session was the duration in seconds spent in each compartment. Mice were then randomly assigned to saline/cocaine conditioning chambers for a total of eight conditioning sessions (cocaine was paired with the less preferred compartment). On each of the four saline conditioning days (Days 1, 3, 5, 7) animals received an intraperitoneal (i.p.) injection of 0.9% NaCl (saline) and were confined to the previously assigned saline-paired chamber for 30 min. For each of the four cocaine conditioning days (Days 2, 4, 6, 8) animals received an i.p. injection of cocaine hydrochloride at a dose of 20 mg/kg (Sigma-Aldrich, Switzerland) at a volume of 5 ml/kg dissolved in 0.9% NaCl− and were confined to the cocaine-paired chamber for 30 min. The preference test (Post Test) was performed 48 hours after the last conditioning day when mice were again permitted free access to all chambers for 30 min. The total time spent in each chamber during the Pre Test and Post Test was automatically recorded for subsequent statistical analyses.

Fat composition and distribution (CT-Scan)
Rodent computerized tomography allows to estimate the volumes of adipose tissue, bone, air, and the remainder, using differences in X-ray density, and distinguishes between visceral and subcutaneous adipose tissue. Measurements were performed non-invasively using a rodent computerized tomography (CT) scanner (LaTheta). Before CT scanning, animals were anesthetized using isoflurane (induction with 4-5% isoflurane in 600 cc O2) and maintained under gas anesthesia (1.5-2.5 % isoflurane in 300 cc O2) throughout the scan, which lasted about 5 min.

Energy expenditure and activity (Metabolic Cages)
Measurements of food and water intake, O2 consumption/CO2 production, were performed in an automatic feeding monitoring system coupled to an open-circuit indirect calorimetry system (TSE Phenomaster System). Mice were single housed under thermoneutral temperature conditions, food and water were available ad libitum and constantly monitored. Each cage was connected to a fresh air supply as well as the sample switch unit for drawing air samples from each cage. Cages (n=12) were enclosed in a ventilated cabinet to precisely control ambient temperature and light. (control, f n=6; control, m n=6; HFD, f n=6, HFD, m n=6).

Insulin tolerance test (ITT)
Mice were food deprived overnight (12h-15h), but not water deprived. In the middle of the dark phase a 5 µL drop of tail blood was taken for baseline glycemia. Blood glucose was measured with a blood glucose monitor and the associated glucose test strips for glucose measurement (Accu-Check Aviva). Mice then received an i.p. injection of insulin (0.75 mU insulin/kg body weight, 10 µL of insulin solution/g body weight) and subsequent blood sampling (5 µL each time point) were collected at 15, 30, 60, 90 and 120 min post injection.

PET quantitative analysis
PET data were analysed with the image analysis software PMOD (PMOD Inc., Zurich, Switzerland) and Microsoft excel. PET images were matched to the CT images and brain regions were delineated according to the mouse brain template implemented in PMOD. The volume of the combined striatum regions was 149 mm 3 . Time-activity curves of brain regions were generated and binding potentials (BP ND ) were calculated by the Logan reference tissue analysis 7 , where t*, the time to reach equilibration was 18 min and correction for k 2 (brain to blood rate constant) was neglected. A sphere in the cerebellum region of 52 mm 3 was used as the reference region 7 . Tracer concentrations in the reference region were calculated from the PET data and the specific radioactivity and scans with > 6.5 nM at 40 min after injection were excluded from the analysis, as receptor saturation with tracer was observed for these scans (data not shown). For each group, 6 scans were finally included in the statistical analysis. PET images of the binding potentials (BP nd ) were generated with the PXMod module of PMOD. The resulting images of the three scans with lowest tracer concentration per group were averaged.

Immunohistochemistry
Adult (PND90) offspring were deeply anesthetized with an overdose of Nembutal (Abbott Laboratories) and perfused transcardially with 0.9% NaCl, followed by 4% phosphate-buffered paraformaldehyde solution containing 15% picric acid. The dissected brains were post fixed in the same fixative for 6 h and processed for antigen retrieval involving overnight incubation in citric acid buffer, pH 4.5, followed by a 90 s microwave treatment at 480. The brains were then cryoprotected using 30% sucrose in PBS, frozen with powdered dry ice, and stored at −80°C until further processing. Perfused brain samples were cut coronally at 30 μm thickness from frozen blocks with a sliding microtome. Six series of sections were collected, rinsed in PBS, and stored at −20°C in antifreeze solution until further processing. For immunohistochemical staining, the slices were rinsed three times for 10 min in PBS, and blocking was done in PBS, 0.3% Triton X-100, 5% normal serum for 1 h at room temperature. The following primary antibodies were used to study dopamine-related markers in various brain areas according to protocols established previously 8 9 .The D1R antibody (D2944) used recognizes 97 amino acids at the C-terminal end of the human D1R and its specificity was previously reported and validated 10,11 . The D2R antibody was raised by immunizing rabbit against a peptide corresponding to 28 amino acid peptide sequence from the human D2R. The D2R antibody has previously been shown to be a specific and reliable marker to label D2Rs 10, 12, 13 . We confirmed it by using the dopamine D2R blocking peptide (AG221; Millipore Bioscience Research Reagents) which consists of 28 amino acid peptide sequence from the human D2R and is manufactured to block the staining of D2R antibody (AB5084P, Millipore Bioscience Research Reagents). Prior to immunostaining, the blocking peptide (50ug/ml) was mixed with D2R primary antibody (10ug/ml) in PBS containing 0.3% Triton X-100 and 2% normal goat serum. As control, only D2R primary antibody (10ug/ml) was added in PBS containing 0.3% Triton X-100 and 2% normal goat serum. Both solutions with or without blocking peptide were then incubated for 2 hours at room temperature. The staining was performed in two identical groups of brain sections, using the blocked antibody for one and the control for the other. Our result clearly demonstrates that the D2R staining observed in the medial prefrontal cortex (mPFC), dorsal striatum and included in the measurement when they came into focus within the optical dissector 16 .

Optical densitometry of dopaminergic markers in prefrontal cortical and striatal regions
Quantification of the immunoreactivity for TH, DAT, D1R, and D2R in striatal regions was achieved by means of optical densitometry using NIH ImageJ software. Optical densitometry was chosen because these dopaminergic markers are highly enriched at synaptic sites in the areas of interest. Digital images were acquired at a magnification of 2.5× (NA 0.075) using a digital camera (Axiocam MRc5; Zeiss) mounted on a Zeiss Axioplan microscope. Exposure times were set so that pixel brightness was never saturated. Pixel brightness was measured in the respective areas of one randomly selected brain hemisphere. In addition, pixel brightness was measured in the corpus callosum (for striatal measures) as background area. The background-corrected optical densities were averaged per brain region and animal. Four to six sections per animal were analyzed in the specimens. All immunohistochemical preparations were quantified in the dorsal striatum [caudate-putamen (CPu)], nucleus accumbens core (NAc core), and nucleus accumbens shell (NAc shell) (see below).

Delineation of brain regions
All brain areas of interest were delineated according to The Mouse Brain in Stereotaxic Coordinates by Franklin and Paxinos 17 . The following brain areas were included in the densitometric and stereological analyses: CPu (bregma +1.34 to +0.14 mm), NAc core and shell (bregma +1.60 to +0.98 mm), SN (bregma −2.80 to −3.64 mm), and VTA (bregma −2.92 to −3.64 mm). Schematic coronal brain sections of the brain areas of interest with reference to bregma are provided in corresponding figure legends.

Post-mortem neurochemistry
Levels of dopamine (DA) and its metabolites (dihydroxyphenylacetic acid, DOPAC; homovanillic acid, HVA) were determined using high performance liquid chromatography (HPLC) according to procedures established before 8,18 . Animals assigned to the post-mortem neurochemical investigations were killed by decapitation on PND 70. HFD and control offspring were killed and dissected in random order. The brains were extracted from the skull within 1 min after decapitation and immediately frozen on dry ice and then were stored at -80 °C until dissection of the brain. For dissection, the frozen brain was placed ventral side up on an ice-chilled plate covered with filter paper and was cut with a razor blade into 1 mm thick coronal sections. The slices were placed on an ice cold dissection plate for the removal of discrete brain regions, using a 1 mm micropunch for the dorsal striatum (dSTR), medial prefrontal cortex (mPFC; including anterior cingulate and prelimbic cortices), nucleus accumbens (NAc; including core and shell subregions), substantia nigra (SN) and ventral tegmental area (VTA). Coronal sections were prepared along the following coordinates with respect to bregma: anterior-posterior +2.3 to+1.3, +1.3 to +0.3, -0.1 to -0.6, -1.2 to -2.2, and -2.8 to -3.8. Tissue punches from the left and right hemispheres of each brain area of interest were combined, weighed and placed in 1.5 ml polypropylene microcentrifuge tubes containing ice cold 300 μl 0.4M HClO4 and homogenized using ultrasound. After centrifugation at 10,000×g for 20 min at 4 ˚C, the clear supernatant layers were removed into a 1 ml syringe and filtered through a 0.2 μm nylon filter to separate the insoluble residue. This solution was immediately frozen and stored at −80 ˚C until injection onto the HPLC system. For all brain regions an aliquot of 20 μl was injected in the HPLC system.

Maternal Behavior
A 2 x 5 x 4 repeated measures ANOVA (treatment x observation sessions per day x 5-day blocks) was employed to assess differences in the 5 categories of maternal behavior.
Nursing behavior: The frequency of occurrence of this posture declined significantly through the lactation period (F3,24 = 4.43; P < 0.02). Control and HFD mothers showed similar changes in the total amount of nursing as evident by the lack of significant treatment x 5-day block interaction.
Licking/grooming: licking/grooming behavior generally decreased toward weaning (F3,24 = 4.43; P < 0.02) and was significantly more frequent in the light phase and licking/grooming occurred at the highest level on block Day 1-5 leading to a significant observations sessions per day x 5-day blocks interaction (F12,96 = 2.03; P < 0.03). There was no significant between control and HFD dams.

| Peleg-Raibstein et al.
Carrying: Carrying decreased constantly across the lactation period as indicated by the significant main effect of 5-day block (F3,24 = 3.27; P < 0.04). No significant difference was detected between control and HFD mothers as was evident by a non-significant effect of treatment, either as a main effect or in interaction with 5-day block.
Mother off pups: out of nest behavior gradually increased during the lactation period leading to a significant main effect of 5-day block (F3,27 = 4.03; P < 0.02). Changes across days did not differ between control and HFD mothers. The main effect of treatment or its interaction with 5-day block did not attain statistical significance. Mothers off pups behavior was significantly more frequent in the dark phase, supported by a main effect of observations sessions per day (F4,36 = 12.22; P < 0.0001).
Passive nursing: passive nursing increased in the first 5-day block, Days 1-5, and then consistently declined as supported by a significant main effect of 5-day block (F3,24 = 8.32; P < 0.0007). There was no significant effect of treatment on passive nursing scores and there was no significant differences between the observation sessions per day.

Mothers' body weight
Mothers' body weights prior to gestation and following 3 weeks of HFD exposure did not show any differences between mothers exposed to HFD and mothers exposed to the control diet (F1,27 = 2.10;
CPP locomotor activity during the condition phase.

SUPPLEMENTARY FIGURE LEGENDS
Supp Fig. 1 The body weights of dams exposed to either control or HFD diets were monitored before mating (a), during middle of gestation (b), after birth (c) and following weaning (d). No difference in body weight was detected between dams exposed to either control or HFD during the 4 different time periods.
Supp Fig. 2 Glucose levels of dams either exposed to control or HFD diets were monitored prior to conception (a), middle of gestation (b), after birth (c) and after weaning (d). There were no significant difference in glucose levels between mothers exposed to HFD and mothers exposed to the control diet.

Supp Fig. 3
Plasma metabolic parameters of dams exposed to either control or HFD diets were examined following weaning. No differences were detected between HFD and control dams in cholesterol (a), triglycerides (b), free fatty acids (FFA, c) and insulin blood levels (d) following birth.
Supp Fig. 4 CT-scan of dams exposed to either control or HFD diet utilized after birth did not reveal any differences in the lean mass (a), total fat, %fat ratio (b), the subcutaneous (c) and the visceral fat (d) between the HFD and control dams. insulin, triglyceride and free fatty acid (FFA) levels were detected between control and HFD offspring ( Fig. 7a-d).
Supp Fig. 7 The intake of normal laboratory chow of HFD and control offspring was monitored in the metabolic chambers. Both offspring born to HFD and control exposed mothers did not differ in consumption of chow diet during the light and dark cycle. Table 1.
Energy balance monitored in the metabolic cages. Both offspring born to HFD and control exposed mothers did not differ in any of the parameters observed during the light and dark cycle: food intake, energy expenditure [VO2 consumption, respiratory exchange ratio (RER)] and physical activity.

Supp Fig. 8
Fat composition and distribution as measured with a computerized tomography (CT-Scan) displayed changes in fat disruption between HFD and control offspring. Maternal HFD treatment led to significantly higher fat ratio (a), higher visceral fat (b) compared to control offspring. Additionally, a trend was detected in subcutaneous adipose tissue in offspring born to HFD mothers compared to controls (c). *p < 0.05; #p = 0.06.
Conditioned place preference (CPP) during each of the eight conditioning days. Offspring from both control and HFD groups were allocated to either saline or cocaine injections over a period of 8 days.