Chronic Consumption of a High-Fat Diet during Pregnancy Causes Perturbations in the Serotonergic System and Increased Anxiety-Like Behavior in Nonhuman Primate Offspring

Animals.

All animal procedures were approved by the Oregon National Primate Center (ONPRC) Institutional Animal Care and Use Committee and conformed to National Institutes of Health guidelines on the ethical use of animals. A complete characterization of the maternal and fetal phenotype has been reported previously (McCurdy et al., 2009).

Adult females.

Briefly, weight- (7–9 kg) and age-matched (5–7 years of age) adult female Japanese macaques (Macaca fuscata) were fed a control (CTR) or HFD for up to 4 years. The CTR diet (Purina Mills) provided 13% of calories from fat, and the HFD (Test Diet; 5A1F; Purina Mills) provided 32% of calories from fat and was supplemented with calorically dense treats. The HFD represents a typical Western diet in regard to the saturated fat content. Monkeys were housed in indoor/outdoor pens in groups of ∼10–12 individuals (male/female ratio, 2/9–10). Monkeys had ad libitum access to food and water. During monthly health checks, animals were examined for pregnancy by palpation, and pregnancies were confirmed by ultrasound (allowing estimate of gestational age).

Fetal offspring.

Fetuses were collected by caesarean section on gestational day 130 (early third trimester; full term is 175 d) from CTR and HFD groups after 2–4 years on their respective diets. Fetal brains (CTR, n = 5; HFD, n = 6) were collected, perfused with 4% paraformaldehyde fixative, and blocked as described previously (Grayson et al., 2006).

Juvenile offspring.

Full-term offspring were maintained on their mothers' diets. Infants and mothers were left undisturbed for the first 30 d after birth. On postnatal day 30, the offspring were weighed and dual energy X-ray absorptiometry scanned to examine body composition. The mother and offspring were then left undisturbed until behavior testing on postnatal day 130. For the CTR group, 4 male and 4 female offspring were examined, and for the HFD group, 12 male and 11 female offspring were examined.

In situ hybridization. Fetal hypothalamic and midbrain blocks were sectioned at 35 μm using a freezing microtome and collected in 1:24 series. Sections were stored in ethylene glycol cryoprotectant at −20°C until use. For in situ hybridization (ISH), 1:8 series of sections were slide mounted in potassium PBS (KPBS), pH 7.4, and vacuum-desiccated overnight. cRNA probes were transcribed from cDNA clones (kindly provided by C. L. Bethea) (Pecins-Thompson et al., 1998) Tryptophan hydroxylase 2 (TPH2; 300 bp), serotonin transporter (SERT; 253 bp), and serotonin 1A receptor subtype (5-HT_1AR; 431 bp), and transcribed in the presence of 100% P³³-labeled UTP (PerkinElmer). Standard ISH methods were used (Grayson et al., 2006). For visualization, probe-labeled sections were exposed to film (Biomax MR; Kodak) for 2 d (SERT) or 5 d (TPH2, 5-HT_1AR). Autoradiographic images were captured using a CoolSnap HQ camera (Photometrics) and MetaMorph software (Universal Imaging). Integrated morphometry analysis was used to measure total density by multiplying total area by optical density of the three levels of the midbrain: rostral (approximately bregma −17.78 ± 1 mm), medial (bregma −19.75 ± 1 mm), and caudal (bregma −23.40 ± 1 mm; reported in relative units). Three or four matched sections were analyzed for each level in each animal.

Fluorescent immunohistochemistry.

Two experiments were performed using standard fluorescent immunohistochemistry methods (Grayson et al., 2010): (1) serotonin (5-HT)-ir projections in sections containing the arcuate nucleus of the hypothalamus (ARH) to determine the terminal field density and (2) TPH2-ir in raphe-containing sections to determine the relative numbers of TPH2-containing neurons. Sections were washed in KPBS and blocked in 2% donkey serum in 0.4% Triton X-100/KPBS. Rabbit anti-5-HT (lot #057K4753, #S5545; 1:5000; Sigma-Aldrich) and rabbit anti-TPH2 (lot number #1068-102, #NB100-74555; 1:2500; Novus Biologicals) primary antibodies were used. The specificity of these antibodies has been validated by preabsorption studies (Sakowski et al., 2006; Dai et al., 2008). Primary antibodies were diluted with 2% donkey serum in 0.4% Triton X-100/KPBS and incubated with the tissue overnight at 4°C. After incubation, the tissue was rinsed in KPBS and incubated in FITC or rhodamine-labeled donkey anti-rabbit or mouse IgG (1:200; Jackson Immunoresearch) in 0.4% Triton X-100/KPBS for 1 h. Sections were wet mounted and coverslipped with glycerol-based mountant and stored at 4°C before analysis.

Image analysis.

Confocal laser microscopy, as described previously (Grove et al., 2000), was used to capture immunofluorescent images. Images were captured with a 25× oil objective (numerical aperture, 0.75). For fluorescence intensity measurements of 5-HT in the ARH, a series of optical planes at 0.5 μm intervals along the z-axis of the section were scanned for each fluorescent signal and stored as a stack of 1024 × 1024 pixel images, processed with MetaMorph and presented as maximum projections totaling 5 μm. Two fields of view per section (right and left) in three equally spaced, anatomically matched sections per animal were imaged and analyzed (CTR and HFD, n = 3). Total immunoreactive fluorescent intensity was measured using the same conditions and threshold parameters for all images. The total gray value was measured for each field. Analysis was performed by individuals blind with respect to group. The MetaMorph Imaging System was used to process the images, and brightness and contrast levels of the digital images were adjusted with Adobe Photoshop (Adobe Systems).

Cell counting.

Immunohistochemical images were captured under fluorescent illumination using a Photometrics CoolSnap HQ camera (Roper Scientific) connected to a Nikon microscope (E800) with a Plan Apo 4× objective. Total numbers of immunoreactive cells were counted in all raphe-containing sections.

Behavioral testing.

Behavior tests commonly used to assess anxious, fearful, and inhibited behavior in young children and NHPs were used (i.e., the human intruder and novel objects tests; file 2001) (Goldsmith and Rieser-Danner 1990; Kalin et al. 1991). On post-natal day 130, each infant and mother were removed from their home pen and placed in a cage located in an adjacent room at 900 h. Ten minutes before the test, the mother was sedated with ketamine HCl (5 mg/kg, i.m.; Fort Dodge Animal Health), and the mother and infant were transported to the behavioral testing suite in a covered transfer box. All behavior tests were initiated and completed between 900 and 1200 h. Upon arrival at the behavioral suite, the infant was hand caught and placed in a standard primate cage in a separate room from its mother. The infant's behavior throughout the tests was videotaped from an adjacent room through a one-way mirror.

Human intruder test.

This test reliably assesses individual differences in primate stress response and anxiety (Williamson et al., 2003) by assessing behavior in three stress-inducing conditions. This test began with a 10 min acclimation period, after which a human intruder (unfamiliar to the monkey) entered the testing room and stood 0.3 m from the cage with their facial profile (a nonthreatening social stimulus) to the monkey for 2 min (profile period). The intruder exited the room leaving the monkey alone for a 2 min control period. The human intruder then reentered the room and made continuous direct eye contact (a threatening social stimulus) with the monkey for 2 min (stare period) before exiting. Behaviors scored included vocalizations, exploration, movement, and response to the human intruder.

Novel objects test.

This test was designed to assess response to a variety of potentially threatening and nonthreatening novel objects. Novel object tests have been used to assess anxiety-like behavior in a variety of species (Belzung and Le Pape, 1994). Latency to examine novelty has been shown to be heritable and stable over time (Williamson et al., 2003), and has been pharmacologically validated (Belzung and Le Pape, 1994). Moreover, evidence in children indicates that decreased latency to explore novel objects or situations is associated with early-onset anxiety (Schwartz et al., 1999).

This test began 2 min after the end of the stare period. The infant was presented with a series of four novel objects. For each object, the human intruder entered the room taking care to avoid direct eye contact, quickly introduced the object, removed the previous object (except for the novel fruit, which was left in the cage), and immediately left the room (within 5 s). Each object was left with the infant for 5 min. The first object to be presented to the infants was a piece of novel fruit (kiwi), which was placed in the cage. After the kiwi, the human intruder then placed a potentially threatening novel object (a commercially available toy, Mr. Potato Head, selected because it has large eyes). Only the eyes and feet were attached to Mr. Potato Head's body to emphasize the eyes. The next object was a colorful hanging bird toy (a nonthreatening novel object), which was hung on the cage. Last, a realistic rubber snake (similar to natural Japanese macaque predators) and a piece of apple (a familiar food) were placed on the tray in such a way that the monkey had to reach across the snake to access the apple. This was done to examine the monkey's willingness to overcome a threatening stimulus (the snake) to get to a desirable object (apple). When the test was complete, the infant was hand caught and placed in the transfer cage with its mother, and both monkeys were returned to their home pen.

The videotapes were scored by an observer blind to maternal diet using the Observer XT software (Noldus Information Technology). The latency (seconds) to intentionally touch each item was scored, as well as behaviors such as movement, exploration, vocalizations, and other responses to the novel objects. We defined latencies to touch the novel object greater than two standard deviations (SDs) above the mean as an anxious response.

CSF assays.

CSF was collected from juvenile offspring at 13 months of age. Samples were snap frozen in liquid nitrogen and stored at −80°C until time of assay. 5-HT and 5-hydroxyindoleacetic acid (5-HIAA) ELISAs were performed by the ONPRC Endocrine Services Core using commercially available kits (5-HT, #RE59121; 5-HIAA, #RE59131; IBL Transatlantic) in 50 μl of monkey CSF according to the manufacturer's specifications (CTR, n = 6; HFD, n = 11).

Statistical analysis.

For all analyses, normality and homogeneity of variance were initially tested. An independent t test was used to compare TPH2, 5-HT_1AR, and SERT mRNA and SERT immunoreactivity in the rostral (rDR) and caudal dorsal raphe (cDR) and CSF 5-HT of CTR and HFD offspring. The latency to touch the novel objects and the number of vocalizations during the acclimation were nonparametric; thus, a Mann–Whitney U test was used to compare CTR and HFD offspring. A univariate ANOVA was used to examine the amount of time spent being active or sedentary. Data are presented as mean ± SEM. Alpha values are considered significant with p ≤ 0.05. Statistical analyses were conducted using the SPSS software package, version 16.0.