Low uric acid levels in patients with Parkinson's disease: evidence from meta-analysis

Figure S5. Transcript levels of (A) 18S rRNA, (B) GAPDH, and (C) β-actin mRNAs in samples fixed with ethanol for 10 s, 30 s, and 60 s in comparison with the untreated samples. Values are expressed as copy number of transcripts per 182 nm2 × 20 μm cryosection thickness. Bars indicate mean ± SEM. One-way ANOVA followed by Tukey post hoc test, where **p < 0.01, ***p < 0.001. n = 7 for the untreated samples, n = 6 for 10 s fixation, and n = 8 for 30 s and 60 s fixation

Additional file: 1 of Application of NeuroTrace staining in the fresh frozen brain samples to laser microdissection combined with quantitative RT-PCR analysis

Figure S1. Visualization of neurons of ethanol-fixed and NeuroTrace-stained third ventricle (D3V) specimen under (A1) a bright field and (A2) a fluorescence radiated field. The staining of the choroid plexus is thought to be a non-specific signal commonly observed in fresh frozen samples stained with regular Nissl stains, such as Cresyl violet, and is often considered negligible as it is irrelevant to the cerebral parenchyma. (A3) Ethanol-fixed hippocampal CA1 region under a fluorescent light, left side with the NeuroTrace stain and right side without it. (B1) Ethanol-fixed and NeuroTrace-stained neurons of the hippocampal DG region (B2) before and (B3) after microdissection, indicated by red arrows. Scales bars: (A) 200 Îźm, (B1) 100 Îźm, and (B2, B3) 25 Îźm

Additional file: 3 of Application of NeuroTrace staining in the fresh frozen brain samples to laser microdissection combined with quantitative RT-PCR analysis

Figure S3. (A–C) Correlation of the transcript levels between the housekeeping genes in unfixed and unstained (Untreated; crosses) and ethanol and NeuroTrace-treated (EtOH/NT; circles) samples: (A) β-actin vs. 18S rRNA, (B) GAPDH vs. 18S rRNA, and (C) GAPDH vs. β-actin. (D–E) Correlation between the transcript levels of the housekeeping genes and Map2: (D) β-actin vs. Map2, (E) 18S rRNA vs. Map2, and (F) GAPDH vs. Map2. Values are expressed as copy number of transcripts per LMD tissue of 182 nm2 × cryosection thickness in volume. For untreated samples, n = 7 for 20 μm, n = 6 for 30 μm, and n = 8 for 40 μm; for fixed and stained samples, n = 7 for 20 μm, n = 7 for 30 μm, and n = 8 for 40 μm

Executive Function Deficits and Social-Behavioral Abnormality in Mice Exposed to a Low Dose of Dioxin <em>In Utero</em> and via Lactation

<div><p>An increasing prevalence of mental health problems has been partly ascribed to abnormal brain development that is induced upon exposure to environmental chemicals. However, it has been extremely difficult to detect and assess such causality particularly at low exposure levels. To address this question, we here investigated higher brain function in mice exposed to dioxin <em>in utero</em> and via lactation by using our recently developed automated behavioral flexibility test and immunohistochemistry of neuronal activation markers Arc, at the 14 brain areas. Pregnant C57BL/6 mice were given orally a low dose of 2,3,7,8-tetrachlorodibenzo-<em>p</em>-dioxin (TCDD) at a dose of either 0, 0.6 or 3.0 µg/kg on gestation day 12.5. When the pups reached adulthood, they were group-housed in IntelliCage to assess their behavior. As a result, the offspring born to dams exposed to 0.6 µg TCDD/kg were shown to have behavioral inflexibility, compulsive repetitive behavior, and dramatically lowered competitive dominance. In these mice, immunohistochemistry of Arc exhibited the signs of hypoactivation of the medial prefrontal cortex (mPFC) and hyperactivation of the amygdala. Intriguingly, mice exposed to 3.0 µg/kg were hardly affected in both the behavioral and neuronal activation indices, indicating that the robust, non-monotonic dose-response relationship. In conclusion, this study showed for the first time that perinatal exposure to a low dose of TCDD in mice develops executive function deficits and social behavioral abnormality accompanied with the signs of imbalanced mPFC-amygdala activation.</p> </div

Compulsive repetitive nose poking in mice exposed to a low TCDD dose (TC-0.6).

<p>(A, B, C) The number of nose pokes per rewarded visit (open triangle) and non-rewarded visit (closed circle): (A) Control, (B) TC-0.6, (C) TC-3.0. Each plotted point indicates the average number of nose pokes per visit made by an individual mouse in a session. (D) Group-averaged numbers of nose pokes throughout the sessions per rewarded visit or non-rewarded visit. Error bars indicate ± S.E.M., n = 8/group. * indicates a significant difference from the Control and TC-3.0 groups (<i>P</i><0.05, ANOVA, followed by Tukey's post hoc test).</p

Impaired behavioral flexibility in TCDD-exposed mice (inter-session analysis).

<p>(A) Overview of IntelliCage apparatus. (B) Group composition of mice housed and tested in each IntelliCage apparatus. (C) Behavioral sequencing task. Mice were allowed to obtain water reward for 4 seconds when they visited an “active” rewarded corner (blue circle). The location of the active rewarded corner was alternately switched between the two diagonally positioned corners each time the mouse received a reward. Thus, the mice had to acquire the behavioral sequence of alternating between the two rewarded corners to continuously obtain rewards. A visit to the never-rewarded corners (gray circles with a diagonal line) was counted as a discrimination error. (D) Serial reversal task. For each mouse, the assigned spatial patterns of the rewarded corners (seq. 1 or seq. 2) were reversed 11 times every 7 or 4 sessions. (E) Time-line of the experiment for each day. (F, G) Learning performance on the behavioral flexibility test. For the purpose of readability, data from the TC-0.6 and TC-3.0 groups of mice were separately plotted in F and G, respectively, whereas the data from the Control group are shown in F and G. Discrimination error rates (the number of discrimination errors in the first 100 corner visits in the session) are indicated as the means ± S.E.M. (n = 8/group). For each session, the individual mouse's discrimination error rate was transformed into a z-score calculated among all the mice. The bars in the insets in F and G indicate the averaged z-scores for each group in the first sessions of all the Revs (early stage of reversal learning) and in the second to fourth sessions of all Revs (late stage of reversal learning). * indicates a significant difference from the Control group (<i>P</i><0.05, ANOVA followed by Tukey's post hoc test).</p

Impaired dendritic growth and positioning of cortical pyramidal neurons by activation of aryl hydrocarbon receptor signaling in the developing mouse

<div><p>The basic helix-loop-helix (bHLH) transcription factors exert multiple functions in mammalian cerebral cortex development. The aryl hydrocarbon receptor (AhR), a member of the bHLH-Per-Arnt-Sim subfamily, is a ligand-activated transcription factor reported to regulate nervous system development in both invertebrates and vertebrates, but the functions that AhR signaling pathway may have for mammalian cerebral cortex development remains elusive. Although the endogenous ligand involved in brain developmental process has not been identified, the environmental pollutant dioxin potently binds AhR and induces abnormalities in higher brain function of laboratory animals. Thus, we studied how activation of AhR signaling influences cortical development in mice. To this end, we produced mice expressing either constitutively active-AhR (CA-AhR), which has the capacity for ligand-independent activation of downstream genes, or AhR, which requires its ligands for activation. In brief, CA-AhR-expressing plasmid and AhR-expressing plasmid were each transfected into neural stems cells in the developing cerebrum by <i>in utero</i> electroporation on embryonic day 14.5. On postnatal day 14, mice transfected <i>in utero</i> with CA-AhR, but not those transfected with AhR, exhibited drastically reduced dendritic arborization of layer II/III pyramidal neurons and impaired neuronal positioning in the developing somatosensory cortex. The effects of CA-AhR were observed for dendrite development but not for the commissural fiber projection, suggesting a preferential influence on dendrites. The present results indicate that over-activation of AhR perturbs neuronal migration and morphological development in mammalian cortex, supporting previous observations of impaired dendritic structure, cortical dysgenesis, and behavioral abnormalities following perinatal dioxin exposure.</p></div

Primer sequences for LMD-RT-qPCR.

<p>Primer sequences for LMD-RT-qPCR.</p

Overexpression of constitutively active-AhR (CA-AhR) reduces the morphological complexity of somatosensory cortex layer II/III pyramidal cell dendrites.

<p>(A) Representative drawings of EGFP-expressing pyramidal neurons in cortical layer II/III of 14-day-old mice. Neurons with the characteristic morphology of CA-AhR, AhR, and control groups were randomly chosen for display (n = 3–4/group). A distinct reduction in dendritic arborization in the CA-AhR group was observed compared to control mice and mice transfected with AhR. (B−G) Quantification of dendritic complexity (B, C), length (D, E), and number of branching points (F, G) of apical (B, D, F) and basal (C, E, G) dendrites. Values are mean ± S.E.M. from 3 control, 3 AhR, and 4 CA-AhR group mice. Asterisks denote significant differences between the control and CA-AhR groups, whereas sharps indicate significant differences between the AhR and CA-AhR groups. Single (*, #) and double (**) symbols denote significance by Tukey–Kramer <i>post hoc</i> test at <i>p</i> < 0.05 and 0.01, respectively. Scale bar = 100 μm.</p

Low competitive dominance in the low TCDD dose (TC-0.6) group.

<p>(A, B, C) Time-course of visit frequency in the first session of the day (21:50–22:40). The gray-colored period indicates the first five minutes (22:00–22:05) of the task. Vertical dotted lines indicate a peak in the group-averaged number of visits. (A, B, C) Time-course of visit frequency at the beginning of the session (21:50–22:40). Colored lines indicate the averaged visit frequency across all the sessions of each mouse, and the black thick lines indicate the average of each group: (A) Control, (B) TC-0.6, and (C) TC-3.0. (D) Visit frequency in the first five minutes throughout the sessions for each group (mean ± S.E.M., n = 8/group). (E, F) Average number of visits and duration of licking per session are shown as indices of daily water consumption. Error bars indicate ± S.E.M., n = 8/group. (G) A diagram of the competition task, in which the Control and TC-0.6 groups of mice (littermates of the mice used in the behavioral flexibility task) were subjected to the same water deprivation schedule as in the behavioral flexibility task. From days 1 to 4, all of the mice (a total of 14 mice, comprised of the Control and TC-0.6 groups) were housed in the same IntelliCage apparatus (a highly competitive condition). From days 5 to 8, each group of mice was housed separately in two different IntelliCage apparatuses (a less competitive condition). From days 9 to 12, all the mice were again housed in the same IntelliCage apparatus (a highly competitive condition). (H) Visit frequency in the first five minutes in the competition task. * and † indicate a significant difference from the Control group in the identical time period and from the TC-0.6 group on days 5 to 8, respectively. Error bars indicate ± S.E.M., n = 7/group.</p