Analytic grade reagents were purchased from IDT Company (San Jose, CA), the Jackson Laboratory (Bar Harbor, Maine), Sigma Aldrich (St. Louis, MO), Cellsignaling Technology (Danvers, MA), Seahorse Bioscience (North Billerica, MA), Invitrogen (Grand Island, NY), and Abcam (Cambridge, MA).

All mice were two months old in the beginning of this study. Among BZDs, we selected lorazepam with an intermediate half-life in human. Lorazepam is currently one of the most frequently used BZDs in clinical settings [41]. Clinicians prefer BZD with an intermediate halflife to extensively sedating long half-life BZDs [42,43]. Lorazepam has been reported to impair motoric function in humans [8] and mice [11]. In the current study, mice were injected with lorazepam (0.5 mg/kg) in the afternoon, and next morning they were tested for motoric function using Rotarod apparatus. This procedure was repeated for 14 days. The dose (0.5 mg/kg) of lorazepam was chosen based on our pilot study and previous studies in which lorazepam was used for anxiolytic effects [44-46]. Rotarod is a motor driven treadmill (Omnitech Electronics, Columbus, OH) that measures running coordination and motor performance, such that a shorter latency to fall from an accelerating rod indicates poorer motor performance. The rotor consists of four cylinders that are mounted 35.5 cm above a padded surface. Mice were placed on the cylinder and a timer switch was simultaneously activated to rotate the cylinders. Acceleration continued until 44 rpm for maximum 90 seconds or animals fell to the padded surface, which simultaneously stopped the timer. Mice were tested for 3 sessions/day for 14 days with a 20 minutes resting period between sessions [47].

Among p38 isoforms, we selected p38α because it is the most abundant isozyme in the brain and the best characterized isoform [48,49]. To avoid lethality, we employed a conditional transgenic mouse system to down-regulate Purkinje p38 genes using the Cre/loxP system and Pcp2 promoter (Purkinje neuron-specific marker) [50,51]. Transgenic mice (Pcp2-Cre mice) that express Cre recombinase under the control of the Pcp2 (Jackson Laboratory) were cross-mated with floxed-p38α mice to generate the Pcp2-Cre^+/−/p38^loxP/loxP mice. The mice with floxed-p38 were kindly provided by Boehringer Ingelheim Inc. The p38 floxed allele was generated by homologous recombination of embryonic stem cells in which two sites of ATG containing Purkinje p38 sequence were flanked by loxP [52,53] and excised in the presence of Pcp2-Cre. When pups were 21 days old, the tips of the tail were collected for genotype identification.

DNA was isolated by incubating tail samples overnight at 55˚C in proteinase K buffer. Primer sequences were as follows: for Pcp2-Cre transgene forward, 5’-GCGGTCTGGCAGTAAAAACTATC-3’; for Pcp2-Cre transgene reverse, 5’-GTGAAACAGCAT TGCTGTCACTT-3’; for Pcp2-Cre internal positive control forward, 5’-CTAGGCCACAGAATTGAA AGA TCT-3’; for Pcp2-Cre internal positive control reverse, 5’-GTAGGTGGAAATTCTAGCATCATCC-3’; loxP-flanked p38α allele: 5’-TCCTACGAGCGTC GGCAAGGTG-3’ and 5’-ACTCCCCGAGAGTTCC TGCCTC-3’. Sequential denaturing (96˚C, 30 sec), annealing (52˚C, 1 minute) and extension (72˚C, 1 minute) were repeated 35 times for genotyping the Pcp2-Cre transgene. The program of 30 cycles of denaturing (94℃ for 30 sec), annealing (58˚C, 30 sec), and extension (72˚C, 45 sec) was used to genotype the p38α alleles using the polymerase chain reaction method [51].

Mice were anesthetized with isoflorane and perfused with 0.9% saline. The formalin-fixed and paraffin-embedded left hemispheres were cut into 8 μm-thick slices on a microtome. The slices were deparaffinized in xylene, rehydrated through a series of graded ethanol solutions, and washed with PBS. The slices were subsequently moisturized at 95˚C and incubated with primary antibody, polyclonal rabbit anti-phosphorylated p38α (pp38, an active form of p38) overnight at 4˚C. The slices were then incubated with broad spectrum poly HRP conjugate for 40 minutes at room temperature. The antigen-antibody bindings were visualized with a diaminobenzidine color reaction for 10 minutes. The slides were further rinsed, dehydrated through a series of graded ethanol and xylene, and mounted with Permount. All photographs were taken with a compatible Zeiss digital camera. A 20- fold magnification was used to take pictures.

Brain slice samples were evaluated using the Carl Zeiss microscope, the image analysis program AxioVision 4 (Carl Zeiss, Thornwood, NY) and a previous method [54] that was modified for our purpose. Three mice per treatment group were evaluated. Six microscopic fields per mouse were selected, such that two microscopic fields were randomly selected from each of anterior (lobes I-V), medius (VI-VII), and posterior (VIII-X) regions of the cerebellar cortex [39,55]. All Purkinje cells with visible p38-positive deposits were individually counted per microscopic field, and the length of the Purkinje layer per field was measured using a software program, Image Pro Plus (Media Cybernetics, Silver Spring, MD) to normalize the cell counts. Data were presented as the average number of p38-positive Purkinje neurons/Purkinje layer (mm) from the 18 data points (3 mice/group, 6 fields/mouse).

Mitochondrial respiration was assessed by measuring mitochondrial O₂ consumption rate (pmoles/minutes) according to a method provided by the XF respirometer manufacturer (Seahorse Bioscience). XF sensor cartridge was hydrated overnight in XF calibration buffer (at 37˚C, no CO₂). Isolated mitochondria were diluted with mitochondrial assay solution (Seahorse Bioscience, North Billerica, MA) to yield a final concentration of 200 µg/ml. Diluted mitochondria (50 µl) were transferred into each well of XF microplate and spun down at 4˚C for 10 - 20 minutes at 2000 - 3600 g. A consistent monolayer of mitochondrial adhesion to well bottom was visually ensured. A volume of 450 µl of succinate (5.5 mM) and rotenone (2.2 µM) was then added to each well. The XF microplate was warmed at 37˚C for 8 - 10 minutes and placed in XF respirometer. Real-time (data are obtained while mitochondria respire) mitochondrial respiration was subsequently recorded every 5 - 7 minutes.

All numerical data are expressed as mean ± standard error of mean (SEM). The data were analyzed by oneway ANOVA as a factor of treatment or two-way ANOVA as a factor of treatment and days of testing. When a significant difference was observed, post-hoc Tukey’s test was conducted to detect an individual group difference. p value was set less than 0.05 to indicate statistical significance.

Figure 1 illustrates the motoric performance of mice that were injected with BZD or vehicle (methyl cellulose) for 14 days. Two-way ANOVA was conducted as a factor of treatment and days of testing. Compared to vehicle-injected mice, BZD-injected mice fell quicker (a shorter latency, poor performance) from Rotarod from day 9 to the end of the test (day 14) (^*p < 0.01). There was no