Amphetamine

All procedures are approved by UCSD or TSRI IACUC committee. C57BL/6 female mice (stock #000664) were purchased from Jackson labs at 9 weeks of age. All mice were fed normal chow until 10 weeks of age. Olanzapine was compounded into high fat diet (HFD) (45 kcal% from fat) at a concentration of 54 mg/kg of diet (OLZ-HFD, Research Diets, Inc., D09092903, New Brunswick, NJ) as used in other studies^{34 (link)–36 (link),81 (link),82 (link)}. Using the power analysis software and our pilot data we calculated the number of mice needed per group [http://www.statisticalsolutions.net]. Control animals were fed HFD (45 kcal % from fat D09092903). The olanzapine dose selected results in mouse plasma levels (21 ± 5 ng/mL) that are similar to levels observed in humans treated with olanzapine (10–50 ng/mL)^{34 (link)}. Minocycline (MINO) was administered in the drinking water at a dose of 0.6 mg/mL, which results in a plasma level of less than ~7 µM, concentration comparable with circulating levels in human patients (standard dosing of 50–100 mg minocycline results in plasma level of 2–11 µM^{83 (link),84 (link)}). At 10 weeks of age mice were randomly divided into four treatment groups (control, OLZ, MINO and OLZ + MINO) and fed these diet/water combinations for the 12 week duration of the study. At 11 weeks of treatment mice were placed in EchoMRI machine to measure fat and lean mass and then placed in Comprehensive Lab Animal Monitoring System (CLAMS) for 3 days to acclimate followed by 2 days of recordings to measure oxygen consumption (V̇O₂), carbon dioxide production (V̇CO₂), respiratory exchange ratio (RER), energy expenditure, total activity at x-axis (XTOT) and total activity at z-axis (ZTOT). At 12 weeks mice were sacrificed and samples snap frozen in liquid nitrogen. Circulating levels of cytokines and gut-derived hormones were measured by multiplex ELISA (lincoplex, st Charles, MO). As a control experiment male C57BL/6 mice (stock #000664) purchased from Jackson labs, were fed either olanzapine-HFD or control HFD from 12 weeks of age for 2 weeks (n = 7 per group). At 14 weeks of age mice were sacrificed and samples snap frozen in liquid nitrogen. Tetracycline co-treatment study: 10 week old female mice were divided into 6 groups (control, OLZ, MINO, MINO + OLZ, TETRA and TETRA + OLZ, n = 6–8 per group). Mice were treated with olanzapine and minocycline as described above, while additional two groups were treated with tetracycline (0.6 mg/ml in drinking water) either alone (TETRA) or in combination with olanzapine (TETRA + OLZ). Food intake and weight gain were measured daily throughout the two-week study. Amphetamine-induced hyper locomotion assay^{38 (link)}. Twelve week old female C57BL/6 mice were divided into 4 groups (control, OLZ, MINO and MINO + OLZ, n = 12 per group). MINO was provided through the drinking water (0.6 mg/mL). One week into these treatments, the mice were habituated to the locomotor activity test (1 hr) following injection of saline (0.9%, IP, 0.01 mL/g), which was repeated again 3 days later. Three days after the second habituation, OLZ (2 mg/kg, 0.01 mL/g, in 1% methylcellulose) or vehicle (1% methylcellulose) was injected IP and the mice were immediately placed in the locomotor activity chambers. 30 min later the mice were removed very briefly from the chamber to receive 2.5 mg/kg D-amphetamine (in 0.9% saline, 0.01 mL/g, IP) and replaced in the system for an additional 120 min. Locomotor activity was measured in polycarbonate cages (42 × 22 × 20 cm) placed into frames (25.5 × 47 cm) mounted with two levels of photocell beams at 2 and 7 cm above the bottom of the cage (San Diego Instruments, San Diego, CA) to record both horizontal (ambulation, center activity and total horizontal activity) and vertical (rearing) behavior every minute for the 30 min pre-amphetamine and 120 min post-amphetamine test phase. Data were analyzed using repeated measures ANOVA followed by Tukey’s multiple comparisons test.

Two weeks after 6-OHDA administration, an amphetamine-induced rotation test was performed to confirm dopaminergic depletion in the nigrostriatal pathway (measure dopamine imbalance). Animals were injected intraperitoneally with amphetamine (4 mg/kg, Sigma-Aldrich). The number of left and right turns was recorded for 90 min using a custom-built computerized image and movement recognition system previously reported by our group [27 (link)]. Animals were evaluated 2 weeks after the 6-OHDA lesion, and only those with >400 ipsilateral turns were selected for transplantation procedures.

Ultrasonic vocalisations were recorded at a sampling rate of 384 kHz with an ultrasonic microphone (sensitivity range: 10 to 160 kHz; M500−384, Pettersson Elektronik, Sweden) attached 10 cm above the cage lid and a freeware sound-recording programme (Audacity 2.1.3).¹ Sound events were identified from spectrograms generated in Audacity with fast Fourier transform (Hanning window of size 1024). USVs were defined as discrete sonic events of peak frequency 20−100 kHz and duration 10−150 ms, with a minimum of 20 ms between 2 events. We chose such broad criteria to be inclusive of any ultrasonic events not previously described in the literature. The current classification of 50 kHz USV subtypes sometimes lack information on frequency, we therefore recorded subtype and estimated frequency independently. For USV subtype identification, we used published USV descriptions [following amphetamine injection, as described in Wright et al. (2010) (link)], and our own experience in detecting USVs emitted during rat tickling or play behaviour (Bombail et al., 2019 (link); Hammond et al., 2019 (link)). When further confirmation was required, the recordings were played at 0.05 × speed to listen to the sounds in the human audible range. To visually assess USV frequency, we estimated the projected median value of the total USV trace onto the frequency axis (to the nearest multiple of 5 kHz). Audacity recordings were visually assessed by two researchers naïve to experimental conditions.
Rats were video recorded during the test meals (Sony 12.0 mega pixels HDR-XR-500 Handycam). Recordings were analysed using a freeware video player that allows frame by frame analysis at the resolution of 40 ms (64-bit PotPlayer).² For each rat, the video and USV track timings were synchronised using the sound of a timer audible on both recordings, at the start of each phase. The timing of this event could be assigned to a video frame (at the resolution of 40 ms) and to the USV spectrogram (to the nearest millisecond). This was used to investigate what actions the rats were performing upon USV production. We also used scan sampling at the resolution of 1 s to describe behaviour over the 10 min of anticipation or food consumption phases (600 observations per individual and phase). The ethogram of all the behaviours detected in the recordings is described in Table 1, it follows criteria based on our previous work (Bombail et al., 2019 (link), 2022 (link); Hammond et al., 2019 (link); Champeil-Potokar et al., 2021 (link)).