Real time PCR analysis of glycine receptor subunits expressed by NPCs after 1 and 3 weeks of differentiation.

<p>Quantitative real-time PCR (SYBR green assay) was performed for each transcript and control (β2-microglobulin). Cycle threshold (Ct) values were normalized to the Ct values of the control and are given as log2^−ΔCt (ΔCt = Ct – Ct β2-microglobulin). Data are presented as means ± S.E.M. of 3 independent experiments (3 NPC lines) each performed in duplicate. A, The bar graph shows predominant expression of α2 and β subunits in NPCs differentiated for 3 weeks that is significantly different from all other glycine receptor subunits; significant differences between log2^−ΔCt values are marked (***p<0.001, ANOVA and Newman-Keuls post test). B, After 1 week of cell maturation the various glycine receptor isoforms are not yet expressed to a significantly different extent. The expression of α2, α4 and β subunits was markedly lower in NPCs differentiated for 1 week than for 3 weeks.</p

Quantitative real-time PCR analysis of glycine receptor subunit expression in human mesencephalic NPCs after 1 and 3 weeks of differentiation.

<p>Primer sequences, amplification product in base pairs, and mean ΔCt values ± SEM (n = 3) are given for each investigated receptor subunit (*p<0.05, t-test). Note, a low ΔCt value represents a high expression level.</p

sEPSP in sMSN from wt and RGS9-deficient mice.

<p>sEPSP in sMSN from wt and RGS9-deficient mice. (<b>a</b>) sMSN with multiple dendrites and a large number of spines, i.e. a typical morphology in wt. (<b>b</b>) Characteristic whole-cell currents showing enhanced sEPSP in RGS9^−/− cells. (c) Example of voltage clamp traces of RGS9^−/− sMSN. (<b>d–f</b>) Quantification of amplitudes (<b>d</b>), inter-event interval (<b>e</b>) and frequency (<b>f</b>) of sEPSPs indicating larger and more frequent sEPSPs in RGS9-deficient animals (*P≤0.05, n = 21 (wt) and 18 (ko), respectively).</p

D2R activation does not inhibit forskolin-stimulated cAMP formation in acute striatal samples.

<p>(<b>a</b>) Forskolin increases cAMP accumulation in acute striatum biopsies from both wt and RGS9-deficient mice with an EC₅₀ of 2.6 μM (n = 4). (<b>b</b>) Acute striatum samples prestimulated with 10 μM forskolin were incubated with the selective D2R agonist quinpirole at concentrations from 10 nM to 100 μM. No reduction in cAMP accumulation was detected at sub-micromolar concentrations. Similar results were obtained when 60 μM forskolin were used for prestimulation (data not shown).</p

Expression analysis using qPCR of selected transcripts involved in LTD, LTP and Ca²⁺ signaling.

<p>Gene expression data are given as 2^−ΔΔC_T ± SEM with the sample size in parentheses. Additionally, the affiliation to LTD, LTP, and the Ca²⁺-signaling pathway is given for each gene. More microarray and qPCR data including non-changed and other significantly changed signaling components are given in Tables S4 and S5 in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0092605#pone.0092605.s001" target="_blank">File S1</a>. *P≤0.05, **P≤0.01, ***P≤0.001.</p

Glycine-induced Ca²⁺ signalling in fura-2 loaded NPCs differentiated for 1 or 3 weeks.

<p>A, Changes in [Ca²⁺]_i were evoked by application of 100 µM glycine (Gly), 100 µM D-serine (D-Ser), 20 µM strychnine (Stry), and 50 mM KCl in cells differentiated for 3 weeks. The trace is the mean response of eight cells from a representative experiment. B, Summary of the [Ca²⁺]_i response amplitudes (n = 22–27 cells; means ± SEM) and fractions of cells (n = 60–75 from 86 cells) responding to different stimuli was obtained from 4 experiments as shown in A. C, NPCs differentiated for 1 week showed smaller increases in [Ca²⁺]_i upon application of 100 µM glycine and 50 mM KCl. The trace is the mean response of 8 cells from a representative experiment. D, Fractions of cells differentiated for 1 week (n = 7–29 of 139 cells) or 3 weeks (n = 60–75 from 86 cells) responding to different stimuli were obtained from 5 and 4 experiments as shown in A and C, respectively.</p

Striatal DARPP32-Thr34 phosphorylation is enhanced in RGS9-deficient mice.

<p>(<b>a</b>) Total DARPP32 from striatal homogenates of 3-month-old wt and RGS9-deficient mice was measured by Western blot quantification and normalized to actin. (<b>b</b>) DARPP32 phosphorylation at Thr34 and Thr75 is given relative to total DARPP32. Representative Western blots are shown, quantification was performed with n = 6–8 per genotype.</p

Immunocytochemistry of human mesencephalic NPCs after 3 weeks of standard differentiation in vitro.

<p>Photomicrographs of NPCs immunoreactive for MAP2 (A) and glycine receptor subunits (B); nuclei were counter-stained with DAPI (C). Merged picture illustrates that neuronal cells express glycine receptor subunits (D). Note, this glycine receptor antibody is specific for all subunits.</p

Striatal ERK1 and ERK2 expression are decreased in RGS9-deficient mice but phosphorylation is increased.

<p>(<b>a</b>) Total ERK1 and ERK2 from striatal homogenates of 3-month-old wt and RGS9-deficient mice was measured by Western blot quantification and normalized to actin. (<b>b</b>) Striatal phospho-ERK1/2 relative to total ERK1/2. Representative Western blots are shown, quantification was performed with n = 7 per genotype.</p

Western blot analyses of key proteins in striata of 3-month-old wt and RGS9-deficient mice.

<p>(<b>a</b>) CaMK IIβ expression (56 kDa) and phospho-CaMK IIβ expression (60 kDa) were not significantly changed in striata of RGS9-deficient mice. Phospho-CaMK IIβ is given relative to total CaMK IIβ. (<b>b, c</b>) CaMK IIγ expression and GluR2 expression were significantly decreased in striata of RGS9-deficient mice. Data were normalized to actin. Representative Western blots are shown, quantification was performed with n = 6 (a, b) and n = 7 (c) per genotype.</p