Analysis of PTZ-induced tonico-clonic seizure in 2n, Ts and Tt mice.

<p>The number of PTZ-treated mice, the number of convulsing mice and the percentage of convulsing mice are given for each genotype and PTZ dose.</p

Generation of a tandem duplication of the <i>Cstb</i> gene on Mmu10.

<p>The targeting vector containing a loxP site (green arrow), a selectable antibiotic resistance gene (neo), and the 5′part of the <i>Hprt</i> gene were integrated in the <i>Cstb</i> locus (<i>Cstbtm1Yah</i>), leading to the tandem duplication of <i>Cstb</i>. The <i>Cstb</i> allele was checked by Southern analysis with probes A and B, and BstXI restriction enzyme, showing a fragment of 9.6 kb for the wild-type allele (wt) and a 11.6 kb fragment (probe A) or a 13.2 kb fragment (probe B) for the <i>Cstb</i> (T) allele.</p

Evaluation of susceptibility of 2n, Ts and Tt mice to PTZ-induced seizure.

<p>(a) Dose-response curves showing the ratio of the number of convulsing mice observed (obs) or predicted (pred) to the total number of injected animals for each PTZ dose. (b) Distributions of latencies of seizure for each genotype at the different doses of PTZ administered. (c) Global survival curves of 2n (blue), Ts (red) and Tt (green) mice (probability of seizure according to time, latencies right censored at 1800 sec).</p

Analysis of <i>Cstb</i> expression in the liver and cerebrum of mice that are disomic (2n), trisomic (Ts) and tetrasomic (Tt) for <i>Cstb</i>.

<p>(a) Real-time PCR analysis: mRNA levels are expressed relative to the disomique control. Data are represented as mean±sem. In both liver and cerebrum, <i>Cstb</i> expression is increased by ∼2 folds in the Ts and by ∼3 folds in the Tt mice. (b) Western blot analysis: band intensities were estimated using ImageJ and normalized against the loading control β-tubulin. Protein levels are represented as fold-changes relative to the 2n control and represented as mean±sem. Amounts of cystatin B are increased by about 1.5 fold in Ts and by about 2.3 fold in Tt brains. Inset shows one representative band for 2n, Ts and Tt.</p

Locomotor activity of 2n, Ts and Tt mice and histological analysis of the cerebellum.

<p>(a) and (b) Mean ± SEM for the total distance travelled and the number of rears during the 30 min session in the open field. (c) Mean ± SEM for the latency to fall from the ratorod. (d) Hematoxylin/eosin-stained coronal sections in through the cerebellum of 6 month-old 2n, Ts and Tt mice (×1,25 and ×40 magnifications) showing similar granular cell layers for the different mice. The position of the enlarged zone in the higher magnifications is shown by the boxes in the top panel.</p

Effects of the dose of <i>Cstb</i> and of the genotype on time of onset of tonico-clonic seizure.

<p>The number of censored mice among the total number of mice, the mean latencies and their standard error estimations for each genotype group and dose of PTZ are presented.</p>§<p>Statistical comparison of genetic groups within each PTZ dose and comparison of doses within each genotype group were done using the log-rank test.</p><p>*Mean latencies and their standard errors estimations were restricted to the largest seizure time among non censored observations.</p

Effect of the dose of PTZ and of the genotype on the probability of seizure (logit scale).

<p>The odds-ratios (OR) are estimated from a logistic regression model. The dose of PTZ is the only statistically significant effect observed. Increasing the administered dose of one mg/kg leads to a significant increase of the probability of seizure. No significant odds-ratio was found when comparing Ts and Tt mice to 2n mice.</p

Effect of a treatment by ifenprodil + cyproheptadine in combination on alcohol induced sensitization.

<p>Y axis: distance travelled in cm (mean ± S.E.M.) A) Initiation of sensitization Session 1: all groups (N = 8 animals per group) received two saline administrations, 15 min and 30 min after being placed in the actimeter. Sessions 2 to 8: groups received saline or ifenprodil 1 mg/kg + cyproheptadine 1 mg/kg and then saline or alcohol (1g/kg ip/session), as indicated in materials and methods. Statistical analyses: repeated measures ANOVA, group effect F_2;21 = 14.2, p< 0.001, session effect F_2;42 = 22.6, p< 0.001, group × session interaction F_4;42 = 9.5, p< 0.001; one-way ANOVAs, differences between groups in session 1 F_2;21 = 2.2, p = ns; in session 6 F_2;21 = 21.7, p< 0.001, in session 8 F_2;21 = 8.3, p< 0.01; post-hoc comparisons, Bonferroni/Dunn test, (a) significantly different (<i>p</i><0.05) from saline group; (b) significantly different (<i>p<</i>0.05) from alcohol group. B) Expression of sensitization: all animals (N = 8 animals per group) received alcohol and no further treatment until session 7. At session 7, animals sensitized to alcohol received ifenprodil (1 mg/kg) + cyproheptadine (1 mg/kg) before alcohol administration (1 g/kg, ip). Statistical analyses: repeated measures ANOVA, group effect F_1;14 = 30.9, p< 0.001, session effect F_2;28 = 19.0, p< 0.001, group × session interaction F_2;28 = 7.6, p< 0.01; Student’s t-test, (a) significantly different (<i>p</i><0.05) from saline group.</p

Effect of ifenprodil (ifen) and cyproheptadine (cypro) in combination on alcohol preference during 2 hour sessions with alcohol (10%) and water bottles.

<p>The preference index Alcohol/Water (Pref.) is calculated as follows: Pref. = (Alcohol consumption–water consumption) / (Alcohol consumption + water consumption). All groups (Saline, N = 12, cypro 1 + ifen 1, N = 8, cypro 3 + ifen 3, N = 9 received saline treatment at the first two sessions, cyproheptadine and ifenprodil treatments started at the arrow. Results are expressed as mean values ± S.E.M. Statistical analyses: repeated measures ANOVA, group effect F_2;26 = 223.2, p< 0.001, session effect F_6;156 = 45.7, p< 0.001, group × session interaction F_12;156 = 9.5, p< 0.001; one-way ANOVAs, differences between groups in session 1 F_2;21 = 0.7, p = ns; in session 6 F_2;26 = 0.1, p = ns; in sessions 7–12, F_2;26≥ 6.1, p< 0.01; post-hoc comparisons, Dunnett test, (a) significantly different (<i>p</i><0.05) from saline group.</p

Effect of prazosin (praz) or cyproheptadine (cypro) separately or in combination on alcohol preference during 2 hour sessions with alcohol (10%) and water bottles.

<p>Doses of cyproheptadine and prazosin were 1 mg/kg and 0.5 mg/kg, respectively. The preference index Alcohol/Water (Pref.) is calculated as follows: Pref. = (Alcohol consumption–water consumption) / (Alcohol consumption + water consumption). All groups (N = 6 animals per group except cypro 1 + prazo 0.5, N = 9) received saline treatment at the first two sessions, cyproheptadine and prazosin treatments started at the arrow. Results are expressed as mean values ± S.E.M. Statistical analyses: repeated measures ANOVA, group effect F_4;28 = 54.8, p< 0.001, session effect F_4;112 = 18.0, p< 0.001, group × session interaction F_16;1112 = 10.0, p< 0.001; one-way ANOVAs, differences between groups in session 1 and 2, F_4;28≤ 1.3, p = ns; in sessions 3–5 F_4;28≥ 9.5, p< 0.001; post-hoc comparisons, Dunnett test, (a) significantly different (<i>p</i><0.05) from saline group.</p