Benzodiazepine (BZD) is the most prescribed CNS depressant in America to treat hyper-excitatory disorders such as anxiety and insomnia. However, the chronic use of BZD often creates adverse effects including psychomotor deficit. In this study, we investigated a novel mechanism by which chronic BZD impedes motoric function in female mice. We used female mice because BZD use is much more prevalent in female than male populations. We tested the hypothesis that the accumulation of p38 (stress-activated protein) in cerebellar Purkinje neurons mediates motoric deficit induced by chronic BZD. To test this hypothesis, we generated transgenic mice that lack p38 incerebellar Purkinje neurons by crossing Pcp2 (Purkinje cell protein 2)-Cre mice with p38loxP/loxP mice. p38-knockdown mice and wild-type mice received BZD (lorazepam, 0.5 mg/kg) for 14 days. During this period, they were tested for motoric performance using Rotarod assay in which a quicker fall from rotating rod indicates poorer motoric performance. Cerebellum was then collected to detect p38 inPurkinje neurons and to measure mitochondrial respiration using immunohistochemistry and real-time XF respirometry, respectively. Compared to vehicletreated mice, BZD-treated mice showed poorer motoric performance, a higher number of Purkinje neurons containing p38, and lower mitochondrial respiration. These effects of BZD were much smaller in p38-knockdown mice. These results suggest that the excessive accumulation of p38 incerebellar Purkinje neurons contributes to motoric deficit associated with chronic BZD. They also suggest that Purkinje neuronal p38 mediates BZD-induced mitochondrial respiratory inhibition in cerebellum. Our findings may provide a new mechanistic insight into chronic BZD-induced motoric deficit.
Benzodiazepines (BZD)s, inhibitory neurotransmitter enhancers, are by far the most frequently prescribed CNS depressants in Americans [
p38 is a signaling protein kinase whose aberrant activation is implicated in many pathological conditions [23-25]. The known members of the p38 family include p38α [
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 [
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+/−/p38loxP/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 [
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 [
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.