Fifty-four (54) adolescent male Lewis rats (postnatal day, PND21) were purchased from Charles River Laboratories (Portage, MI). The rats were grouped (2 per cage) and fed ad libitum. Rats were fed purified diets based on standard AIN-93G formulations (7 g% fat; mainly from soybean oil). The rats were kept under standard housing conditions (22 ± 2°C and regular 12 h dark/light cycle). The diet was obtained from Bio-Serv (Frenchtown, NJ, USA; Cat# F06405) and is based on standard AIN-93G formulations (%kcal from carbohydrates: 64.7; %kcal from protein: 18.8; %kcal from fat 16.5; 3.77 kcal/g). Anderson's Bed O' Cob 1/8’’ was used as bedding material (Newco Specialty, Rancho Cucamonga, CA; Item number 93009). The rats were handled for 5 days before behavioral testing. Behavioral tests were conducted between 10:00–14:00 h.
Elevated Plus Maze (EPM)
The rats were tested on the EPM at PND 130. The near infra-red-backlit EPM consisted of two opposite open arms (50.8×10.2 cm) and two enclosed arms (50.8×10.2×40.6 cm) elevated 72.4 cm above the floor (MedAssociates, St. Albans, VT, USA) The junction area between the four arms measured 10×10 cm. Behaviors were recorded in a dark room. The rats were placed on the central platform facing an open arm at the beginning of each 5 min trial. The maze was cleaned after each test session. Arm activity (total number of entries and duration in each arm) was determined using Ethovision® XT tracking software. From this data, we calculated the well-validated anxiety index according to Cohen et al., and Contreras et al.,:
Anxiety Index= 1 − [([cumulative duration in open arm / total test duration] + [number of entries in open arm / total number of entries to closed arms + total number of entries to open arms]) / 2].
The acoustic startle reflex (ASR) was performed using the SR-Lab Acoustic Chambers (San Diego Instruments). An experimental session began with a habituation program, consisting of 5 min of background noise at 68 dB. Following habituation, the initial block consisted of 11 acoustic startle trials was recorded (120 dB; 20 ms). The trials were presented in a randomized manner (intertrial interval = 10–25 s). The second block had 36 trials presented in a pseudorandomized order. In this block, the trials included pulse-alone trials (120 dB) and pre-pulse trials (5 trials each: 71, 74, or 80 dB; 40 ms duration). The third block of the session consisted of 5 additional pulse-alone startle trials (120 dB). Subsequently, the rats were returned to their cages and each chamber was cleaned with soap and water and thoroughly dried. Max startle amplitudes during the session were averaged.
Modified Light-Dark Test: Mechanical Conflict-Avoidance System
Conflict-avoidance behaviors were measured using the Mechanical Conflict-Avoidance System (MCS) (Noldus). The MCS consisted of one light and one dark chamber (16.5 cm wide × 21.5 cm deep × 15.25 cm high, each) connected by an enclosed alleyway chamber (39.5 cm wide × 21.5 cm deep × 15.25 cm high). All 3 chambers were constructed from acrylic resin and colored red. An array of stainless-steel sharp nociceptive probes (tip dimension = 0.4 mm) was embedded below the floor of the alleyway chamber. The probe array was lowered below the alleyway floor for training sessions and elevated for testing. In this system, anxiety-like behaviors are expressed as decreased escape latency from bright light to the dark compartment. The MCS protocol consisted of 5 days. We used a dim red-light lamp for the whole duration of the experiments. During the first two days, the rats were acclimated to the MCS. The light of the device was turned off and the rats placed in the light chamber (LC). After 10 sec, the aversive bright light was turned on and remained on for 20 sec. Afterward, the LC guillotine door was carefully opened and the rats allowed to explore the MCS for 4.5 min (total duration of each acclimation session was ~5 min). The experimental sessions took place on days 3 to 5. During each testing session, different probe heights were used on each day (0 mm for day 3, 1 mm for day 4, and 3 mm for day 5). For testing days, the light of the device was turned off and the rat was placed in the LC. After 10 sec, the light was turned on and remained on for 20 sec. The LC door was carefully opened and the time to exit the LC was recorded. Each rat was given 30 sec to exit LC which was counted as the latency time. After the rats spent 5-10 sec in the dark chamber, the lid was removed and the rats were returned to their home cages. The apparatus was clean thoroughly between each test. For each experimental session, we repeated this process 3 times per rat with at least 10 min between trials.
Sensory thresholds to a mechanical stimulus were measured using an electronic von Frey aesthesiometer (IITC Life Science, Woodland Hills, CA). Each rat was placed in a Plexiglas chamber on top of an elevated mesh stand that provided access to the plantar surface of the paws. The rats were allowed to acclimate to the testing chamber for 30 min prior to testing. 3 mock trials were performed halfway through the acclimation period. Subsequently, a rigid blunt tip that was attached to the aesthesiometer was applied to the plantar surface. The withdrawal threshold or mechanical sensitivity was defined as the average force (g) required for paw withdrawal in 5 trials separated by at least 2 min interval. The maximum and minimum threshold values were excluded from each paw before analyses.
Intraperitoneal glucose tolerance test (IPGTT)
The IPGTT was performed at PND 170. The rats fasted for 12 h prior to being anesthetized with pentobarbital (100 mg/kg). Subsequently, the rats received intraperitoneal injections of 40% dextrose solution (2 g of dextrose per kg of body weight;). Blood was sampled at 0, 30, 60, 90, and 120 min for glucose analyses. Glucose levels were measured using a glucometer (OneTouch UltraMini® manufactured by LifeScan, Inc Milpitas, CA, USA).
Measuring corticosterone and leptin concentrations using ELISA
The rats were placed on new bedding 24 h before feces collection. Fecal samples were collected from each cage at PND 170 and corticosterone extracted following previous methods. Briefly, fecal corticosterone was extracted by incubating feces in ethanol overnight at a ratio of 10 mL ethanol per gram of feces. Corticosterone concentration was determined using the Correlate EIA kit (Assay Designs, Ann Arbor, MI). ELISA plates were read at 405 nm (Qcal Biotek Instruments plate reader, Winooski, VT USA). Specific concentrations for each sample were determined as percentage bound using a standard curve of samples ranging from 32 to 20,000 pg/mL. Values were then calculated and reported as picograms of corticosterone per mL.
Leptin plasma levels were measured using a commercial ELISA kit (Abcam). Plasma samples were diluted with kit assay buffer (1:3 dilutions). ELISA plates were read at 450 nm on a plate reader (Molecular Devices). Specific concentrations for each sample were determined as mean absorbance using a standard curve of samples ranging from 0 to 8000 pg/mL.
2-AG was extracted from plasma according to published methods. Blood was collected by cardiac puncture and stored in tubes with EDTA, then plasma was obtained by centrifugation (1500 g for 10 min, maintained at 4°C). All samples were stored at -80°C until processing, at which time plasma (0.1 mL) was added to 1.0 mL of a methanol solution containing the internal standard, [2H5] 2-AG (Cayman Chemical, Ann Arbor, MI, USA). Lipids were extracted with chloroform (2 mL) and washed with 0.9 % saline(0.9 mL). Organic phases were collected and separated by open-bed silica gel column chromatography as described, then dried under N2 stream (99.998% pure) and resuspended in 0.1 mL of methanol: chloroform (9:1). Quantitation of 2-AG levels was made by ultra-performance liquid chromatography coupled to tandem mass spectrometry (UPLC/MS/MS), detailed below.
Measurement of 2-AG
Data was collected using an Acquity I Class UPLC system coupled to a Xevo TQ-S Mass Spectrometer (Waters, Milford, MA, USA) with accompanying electrospray ionization (ESI) interface. Lipids were separated on an Acquity UPLC BEH C18 column (2.1×50 mm i.d., 1.7 µm, Waters) with inline Acquity guard column (UPLC BEH C18VanGuard Pre-column; 2.1×5 mm i.d., 1.7 µm, Waters), and eluted by a gradient of methanol in water (0.25% acetic acid, 5 mM ammonium acetate) according to the following gradient at a flow rate of 0.4 mL per min: 80% methanol 0.5 min, 80% to 100% methanol 0.5–2.5 min, 100% methanol 2.5–3 min, 100% - 80% methanol 3–3.1 min). The column temperature was maintained at 40°C, and samples were maintained in the sample manager at 10°C. Argon (99.998%) was used as the collision gas. MS detection was in positive ion mode and capillary voltage set at 0.1 kV. Cone voltage and collision energy as follows, respectively: 2-AG = 30 v, 12 v; [2H5] 2-AG = 25 v, 44 v. Lipids were quantified using a stable isotope dilution method detecting protonated adducts of the molecular ions [M+H]+in the multiple reaction monitoring (MRM) mode. Extracted ion chromatograms were used to quantify 2-AG (m/z= 379.3 287.3) and [2H5] 2-AG (m/z = 384.3> 93.4), which was used as an internal standard.
Statistical analysis was performed using GraphPad Prism 8. We used the K-means clustering method to assign all rats to two groups according to their anxiety index (SPSS Version 25.0; IBM Corp., Armonk, NY). Statistical differences were evaluated using 2-tailed unpaired Student’s t-test or 2-way ANOVA with multiple comparisons, as indicated. P values of less than 0.05 were considered statistically significant.