Supplementary online material
Animals
Female C57BL/6J mice (bred and maintained at the Franklin Wilkins BSU facility, KCL) were fed a palatable obesogenic diet (16% fat, 33% sugar) for six weeks prior to gestation. The diet was then continued throughout gestation and the offspring subsequently weaned onto standard chow, as previously described (Samuelsson et al, 2008). Litters were reduced to 3 males and 3 females at 48 hours to standardize liter size and milk supply. All offspring were weaned onto standard chow and group housed by gender. Male offspring of obese mothers (OO; n=9) were compared to control male offspring (OC; n=8) of mothers fed standard chow (one mouse per litter). OO and OC mice were transferred when aged between 3-4 months and maintained in the Biological Services Unit (BSU) at the Institute of Psychiatry, London, U.K. All housing and experimental procedures were performed in compliance with the UK Home Office Animals Scientific Procedures Act 1986 and according to the protocols approved at King’s College London.
The mice were individually housed in standard cages (40×25×12 cm) with food (Rat and Mouse No. 1 Diet, Special Diet Services, Essex, UK) and water available ad libitum. The housing room was maintained at constant room temperature (21 ± 2 °C) and humidity (45%) and kept under a regular light/dark schedule with lights on from 08:00 am to 20:00 hours. Sawdust (Litaspen premium) and nesting materials (Sizzlenest, Datsand, Manchester, U.K.) in each cage were changed once every two weeks, but never on the day before, or the day of, testing to minimize the disruptive effect of cage cleaning on behavior.
Procedures and analysis
Adult mice aged between 4-5 months were tested through a battery of activity (homecage), anxiety (open field and light/dark box), and simple neurological screens (elements from the primary screen of SHIRPA). Behavioral tests in the battery were performed in the order homecage, open field, light/dark test, homecage and SHIRPA to ensure that the tests considered to be least stressful were performed first. All tests were performed during the light cycle between 09:00 and 19:00 hours with the exception of the homecage task when behavior was also recorded for an hour during the middle of their dark phase. Mice were allocated a new ID number when arriving at the Institute of Psychiatry, London, U.K, and to ensure that the experimenters were blind to their group allocation and mice were tested in a pseudorandom order to avoid test order biases. All mice were allowed to habituate for 2 weeks before undergoing the battery of behavioral tests.
Mice were moved to the testing room immediately before testing and returned as soon as the test was completed. Following each test, fecal boli and urine were removed from the equipment and the equipment was wiped clean with 1% Trigene® solution to avoid olfactory cueing influencing behaviors. During each test the behavior of the mice was recorded on videotapes and then later analyzed using EthoVision software (Noldus Information Technologies bv, Wageningen, The Netherlands; http://www.noldus.com/site/doc200403002). Body weights of the mice were taken before, during, and after testing (see Supplemental Table 1). Organ and fat pad weights are shown in Supplemental Table 2
The open field test (1) and light dark box (2) were used to measure anxiety-like behaviors in the mice. Each test measures the conflict between rodents’ exploratory behavior and aversion to open and brightly lit areas.
Open field
The open field was made of white acrylic, with internal measurements 72 x 72 x 33 cm. White light at a level of 25 lux was evenly distributed across the arena during the testing. Each mouse was placed individually into the corner of the open field, facing an outer wall, and the trial recorded via a camera placed above the open field. Each animal was allowed to freely explore the open field for 10 min, after which the mouse was returned to its homecage. Using EthoVision software, a virtual central square (measuring 26 x 36 cm) was defined and called the ‘central zone’. The remainder of the open field outside the central zone was defined at the ‘outer zone’. Anxiety parameters measured included number of entries into, and time spent (s) in, the central zone of the area. Activity parameters measured were mean speed (cm/s) and distance traveled (cm) in the outer and central zones, as well as the whole of the area.
Light/dark box
The box was made of white acrylic and measured 44 x 21 x 21 cm. The box was divided into two chambers; a smaller dark chamber (20 lux) that occupied roughly 1/3 of the total box, and a larger light chamber (80-110 lux) which was lit with a bright white light. There was a small opening (5 x 7cm) in the separator wall between the two chambers which allowed the mice to freely move between the light and dark compartments. A camera was positioned above the light/dark box, which was connected to a video recorder. The task ran for 5 mins, and started after the mouse was placed into the dark chamber. Following completion of the task, the mouse was returned to its homecage. Activity measures (mean speed [cm/s] and distance traveled [cm]) in the light and dark compartments were derived from the EthoVision tracks and the anxiety parameters, the number of entries into, and time spent in, the light compartment and number of rears in the dark, were hand-coded from the videotape by an experimenter blind to the experimental group.
Homecage activity
The homecage task aims to study the spontaneous locomotor activity of mice in a novel and habituated environment. Spontaneous activity was measured in a familiar homecage-like situation for three one-hour periods, over a day (3).
The spontaneous activity cage was adapted from the standard mouse housing cage (Macrolon type II cage; 40 x 25 x 12 cm) by moving the food hopper and water bottle to the side of the cage, which gave ad libitum access from inside the cage and placing a transparent Plexiglas lid on the top to prevent the mouse from escaping. The test room environmental conditions (light cycle, temperature and humidity) matched those of the mouse housing room. Over one 24-hour period, six spontaneous activity cages were run together. At the start of the 23rd hour test period, an even amount of sawdust (Litaspen premium) was added to a clean activity cage but nesting material was not used as it would interfere with the background image used by the tracking system. Seven red multi-LED cluster lamps (LED cluster red light No. 310-6757; RS Components Northants, UK) of approximate wavelength 705 nm were placed in the test room to provide sufficient lighting for the image capturing, but give the appearance of darkness to the mice given the wavelength of the lamps. A camera, mounted 1 meter above the cages, was connected to a video recorder. The activity of the mouse in each cage was recorded for one hour immediately after transfer (10.30-11.30am; novelty hour), an hour in the middle of their dark phase (1-2am; dark hour) and an hour 22 hours after transfer (8.30-9.30am; habituation hour).
Mean speed (cm/s) and distance traveled (cm) in the homecage were derived from the Ethovision tracks. These measures were assessed in 10-min time bins to examine possible temporal patterns of activity across each hour.
Primary screen of SHIRPA
An observational assessment of each mouse was obtained using a subset of items selected from the primary screen of SHIRPA (Smithkline Beecham Pharmaceuticals; Harwell, MRC Mammalian Genetics Unit; Imperial College School of Medicine at St Mary’s; Royal London Hospital, St Bartholomew’s and Royal London School of Medicine; Phenotype Assessment; 4). SHIRPA is a well-established procedure consisting of standardized protocols, to screen for potential impairment in neuromuscular, sensory, and autonomic functioning. Each mouse was placed into a transparent acrylic cylinder, height and diameter 13cm, for 1 minute for general observation. A black acrylic sheet, 20x14.5cm, was then used to transfer the animal into the arena. An arena (53 x 36 x 18 cm), floor was divided into a grid of 10x10cm squares and was used to determine the locomotor activity of the mice by counting the number of squares crossed within 30 seconds following transfer. Subsequently, the following sensorimotor reflexes were assessed: gait (0-3; normal to incapacity), pelvic elevation when walking (0-3; flattened to elevated 3 mm), startle response (0-3; jerk in response to a sound from a click box), touch escape (0-3; none to extremely vigorous), trunk curling (absent or present), visual placing (0–4; forepaw paddling to none), grip strength (0–4; none to strong), wire maneuver (0–4; active and grips with hind paws to falls off immediately), negative geotaxis (0–4, movement on a grid raised to the vertical). The SHIRPA screen indicated that all mice functioned normally.
Blood pressure and heart rate variability
Measures of blood pressure and heart rate were collected via radiotelemetry in littermates at 3 months of age (n=6). For a description of the methods used, refer to Samuelsson et al (5, 6).
Statistical Analysis
Data analysis was performed using Statistica 7.1 (StatSoft Inc, Tulsa, USA). For the open field, light/dark box and body weight data, groups were compared using two-tailed Student’s t-test. The homecage data were analyzed using a between/within ANOVA (between factor of group and within factor of time bin). Group comparisons at specific time bins were made using Student’s t-test. Data from SHIRPA were analyzed using Mann Whitney U test.
Supplemental References
1. Hall CS. J of Comparative Psychology 1934; 17: 89-108.
2. Crawley J, Goodwin FK. Pharmacol Biochem Behav. 1980; 13; 167-170.
3. Lad HV, Liu L, Paya-Cano JL, Parsons MJ, Kember R, Fernandes C, Schalkwyk LC. Physiol Behav. 2010; 99: 301-316.
4. Rogers DC, Fisher EM, Brown SD, Peters J, Hunter AJ, Martin JE. Mamm Genome. 1997; 8: 711-713.
5. Samuelsson, AM, et al. Hypertension. 2008; 51(2): 383-92.
6. Samuelsson AM, Morris A, Igosheva N, et al. Hypertension. 2010; 55: 76-82.
Supplemental Table 1. Body weight of, and behaviors made by, control (OC) or the offspring of obese mothers (OO) in the open field, light/dark box, homecage (second and third hours of recording: dark and habituated hours respectively) and SHIRPA. * p<0.05, ** p<0.01; two tailed Student’s t-test; # p<0.05; Mann Whitney U.
Supplemental Table 2. Organ weights, systolic blood pressure (SBP) and heart rate variability (low-frequency [LF] component to high-frequency [HF] component ratio) of control (OC) or offspring of obese mothers (OO) at 3 months of age, n=6. * p<0.05; Mann Whitney U.