Sodium current measurements in control and LQTS-3 cells.

<p>Voltage-gated Na⁺ channel currents were recorded in a voltage-clamp mode. Values for current density (A), time of 90% inactivation (B) and time to peak (C) are shown for human ES cell derived CM (n=8), control iPS cell-derived CM (n=11) and for LQTS-3 iPS cell-derived CM from patients NP0012 (p.R535Q, n=13) and NP0016 (p.V240M, n=12). (D,E) Exemplary current traces with an increased persistent sodium current typical of LQTS-3 in diseased R535Q-LQTS-3 CM but not in healthy ES cell-derived CM. Symbols: # p<0.05, * p<0.02, ** p<0.01, ***p<0.005. Differences between all other groups were not statistically significant.</p

Characterization of LQTS-3 iPS cells.

<p>V240M-LQTS-3 iPS cell colony derived from patient NP0016 showing typical human ES cell-like morphology (A) and positive staining for alkaline phosphatase (B). The V240M-LQTS-3 iPS cell colonies expressed pluripotency markers OCT4, SOX2, NANOG, SSEA4, TRA-1-80 and TRA-1-60 as determined by flow cytometry (C) and immmunostaining (D). Expression of pluripotency markers in V240M-LQTS-3 iPS cells by qRT-PCR. Expression values were normalized to GAPDH and are presented as mean +SEM (n=3) relative to corresponding transcript levels in human ES cells (E). Methylation levels of promoter regions of OCT4 and NANOG genes in three different clones of R535Q-LQTS-3 iPS cells at passage 7 and R535Q-LQTS-3 fibroblasts (F). The LQTS-3 iPS colonies derived from dermal fibroblasts of patient NP0012 were verified for the presence of a heterozygous <i>SCN5A</i> point mutation p.R535Q by DNA sequencing (G).</p

Patch clamp recordings of action potentials in R535Q-LQTS-3 iPS cell-derived CM.

<p>(A) Overlay of trace from R535Q-LQTS-3 and healthy cells representing the difference in action potential duration at 50% of repolarization (APD50) in ventricular (left) and atrial (right) like cells. (B) Statistical analysis of the differences in APD90 and APD50 of control and diseased cells. </p

Expression of cardiac markers in R535Q-LQTS-3 iPS cell-derived CM.

<p>(A) Immunocytochemical analysis of cardiac markers α-actinin (red) and troponin I (green). Nuclei were counterstained with DAPI (blue). Scale bars: 20 μm (α-actinin) and 100 μm (troponin I). (B) The analysis of expression levels of cardiospecific transcripts was done by RT-PCR showing that the R535Q-LQTS-3 iPS cell-derived cardiomyocytes express cardiac transcripts at a similar level as their human ES cell-derived counterparts.</p

Early-life adversity selectively impairs a2-GABAA receptor expression in the mouse nucleus accumbens and influences the behavioral effects of cocaine

Haplotypes of the Gabra2 gene encoding the a2-subunit of the GABAA receptor (GABAAR) are associated with drug abuse, suggesting that a2-GABAARs may play an important role in the circuitry underlying drug misuse. The genetic association of Gabra2 haplotypes with cocaine addiction appears to be evident primarily in individuals who had experienced childhood trauma. Given this association of childhood trauma, cocaine abuse and the Gabra2 haplotypes, we have explored in a mouse model of early life adversity (ELA) whether such events influence the behavioral effects of cocaine and if, as suggested by the human studies, a2-GABAARs in the nucleus accumbens (NAc) are involved in these perturbed behaviors. In adult mice prior ELA caused a selective decrease of accumbal a2-subunit mRNA, resulting in a selective decrease in the number and size of the a2-subunit (but not the a1-subunit) immunoreactive clusters in NAc core medium spiny neurons (MSNs). Functionally, in adult MSNs ELA decreased the amplitude and frequency of GABAAR-mediated miniature inhibitory postsynaptic currents (mIPSCs), a profile similar to that of a2 “knock-out” (a2-/-) mice. Behaviourally, adult male ELA and a2-/- mice exhibited an enhanced locomotor response to acute cocaine and blunted sensitisation upon repeated cocaine administration, when compared to their appropriate controls. Collectively, these findings reveal a neurobiological mechanism which may relate to the clinical observation that early trauma increases the risk for substance abuse disorder (SAD) in individuals harbouring haplotypic variations in the Gabra2 gene

Respiratory pattern and apnea in <i>Ndn</i>−/− young adult versus <i>Ndn</i>+m/−p mice.

<p>While the mean apnea duration was similar between genotypes (∼900 msec), evaluation of the total apnea duration expressed as the total recording time (i.e. apnea scores) revealed a two-fold increase in the percentage of total apnea duration in <i>Ndn−/−</i> compared to <i>Ndn+m/−p</i> individuals.</p><p>Values are represented as Mean±SD; n = 20 for <i>Ndn+m/−p</i> and 18 for <i>Ndn</i> −/− mice.</p><p>Mann Whitney t-test, two-tailed. P value = 0.02 (a) and 0.03 (b).</p

<i>Ndn</i> expression in <i>Ndn+m/−p</i> E12.5 embryos.

<p>Expression of <i>Ndn</i> in the nervous system of WT and <i>Ndn+m/−p</i> embryos at E12.5 revealed by IHC or ISH on frozen sections using a Necdin specific antibody (Ndn,red) or <i>Ndn</i> RNA probe (green). Tissue sections are visualized using a Hoechst labeling (blue). Expression is detected in both genotypes, at the protein and transcript levels, in the preoptic area (A), supraoptic area (B), thalamus (C), pons (D) and in the dorsal root ganglia (E). Note that the level of expression is weaker and more restricted in <i>Ndn+m/−p</i> embryos. Other structures, like the tegmentum, the subthalamus and the spinal cord also express the <i>Ndn</i> maternal allele. Scale bar: 50 µm.</p

<i>NDN</i> expression in PWS patients.

<p>Detection of <i>NDN</i> transcripts revealed by ISH, and using a <i>NDN</i> anti-sense probe, on PVN brain sections from control individuals (A,C,E) and PWS patients (B,D,F). A <i>NDN</i>-sense probe was used as a negative control. IHC on SON brain sections, using a NECDIN specific antibody, was performed on the same control and PWS patients. The expression was studied in the 94-118 adult control male (A) and the 95104 adult PWS patient with a maternal uniparental disomy (B), in the 88-017 adult control male (C) and the 00-028 PWS adult patient with a deletion (D), in the 97-153 control infant (E) and the 99-079 PWS infant with a deletion (F). Scale bar: 20 µm.</p

<i>Ndn</i> expression in the 5HT raphe nuclei of <i>Ndn+m/−p</i> individuals.

<p>(A) % of Necdin immunoreactive cells among 5HT positive cells located in the B1 to B9 raphe nuclei. Wild-type (n = 3), <i>Ndn</i>−/− (n = 1) and <i>Ndn+m/−p</i> (n = 10) newborn mice were analyzed. (B) Number of 5HT-expressing neurons in the B1/B2 raphe nuclei of +/+ (n = 9), <i>Ndn +m/−p</i> (n = 18) and <i>Ndn−/−</i> (n = 8) individuals. <i>Ndn+m/−p</i> (n = 18) individuals are divided in two populations: a population in which Necdin immunolabeling is detected in a mean of 46% of 5HT neurons of <i>Ndn+m/−</i> (Ndn+, n = 9) individuals and a population in which no Necdin/5HT colabelling is detected in the B1/B2 raphe nuclei of <i>Ndn+m/−p</i> (Ndn−, n = 9) individuals. Scale bar: 10 µm.</p

<i>Ndn</i> expression analyzed by RT-qPCR in <i>Ndn+m/−p</i> mice.

<p>RT-qPCR analysis of <i>Ndn</i> transcripts. A) <i>Ndn</i> transcripts in E12 WT embryos and selected brain tissues from P1 (whole brain, hypothalamus, pons) or adult (hypothalamus) WT mice. B) <i>Ndn</i> transcripts in <i>Ndn+m/−p</i> E12 embryos and brain tissues from P1 (whole brain, hypothalamus, pons) or adult (hypothalamus) <i>Ndn+m/−p</i> mice. C–F) Quantification of <i>Ndn</i> transcripts in <i>Ndn</i>+m/−p E12 embryos (C,D) and whole brain (C,E) and pons (C,F) from P1 mice with respect to parental genotype (C) and to the maternal or paternal genotype contribution indicating a grandparental influence (D–F). G) Quantification of <i>Ndn</i> transcripts in <i>Ndn+m/−p</i> E12 embryos, P1 and adult <i>Ndn+m/−p</i> brain tissues in male (M) and female (F) mice. H) Quantification of <i>Ndn</i> transcripts in <i>Ndn+m/−p</i> E12 embryos and <i>Ndn+m/−p</i> P1 mice in C57Bl/6J and S129Sv/Pas mouse strains. The <i>Ndn</i> transcript copy number in <i>Ndn+m/−p</i> offspring (E12, P1 brain, P1 pons; n = 71) issued from a cross between a WT female and a <i>Ndn</i>+/− male is significantly more than 2 fold higher than the <i>Ndn</i> copy number in <i>Ndn+m/−p</i> offspring (n = 51) issued from a <i>Ndn+/−</i> female crossed with a <i>Ndn−/−</i> male (C). Considering separately the effect of the maternal or paternal genotype, we showed that when the mother is WT and the father is (+/−), with a maternal <i>Ndn</i> mutant allele (−m/+p) or a paternal <i>Ndn</i> mutant allele (+m/−p), then there is no difference in the copy number of <i>Ndn</i> maternal transcripts between the <i>Ndn+m/−p</i> individuals of the offspring of both types of crosses (n = 25 and n = 46, respectively) (D,E,F). However, we observed an effect of the maternal genotype, with a significant difference in the level of <i>Ndn</i> maternal transcripts between the <i>Ndn+m/−p</i> individuals (n = 22) issued from a (−m/+p female X −/− male) compared with the <i>Ndn+m/−p</i> individuals (n = 29) issued from a (+m/−p female X −/− male) (D,E,F); the +/+ or <i>Ndn+m/−p</i> maternal genotype is correlated with a significant three times higher level of <i>Ndn</i> maternal expression in the <i>Ndn+m/−p</i> offspring. Values are represented as Median (Q1, Q3). WMW test, two-tailed. * P value<0.05 and ** <0.01.</p