JCS033464 Supplementary Material

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• Supplemental Figure S1 -

  Fig. S1. Localisation of endogenous DA in mixed cultured hippocampal neurons. (A,B) Developmental changes of DA distribution in hippocampal neurons. DA is expressed within the cell body and dendrites at 7 (A) and 21 DIV (B). Although the immunostaining is more diffuse at 7 DIV (A, inset), at 21 DIV, the immunostaining appeared punctate (B, inset). Scale bars: 5 µm in A and B; 1 µm in insets. (C-E) DA does not colocalize with synaptophysin but colocalizes with PSD-95 and F-actin at dendritic spines of hippocampal neurons. 21-day-old cultured hippocampal neurons were immunostained simultaneously for DA and either synaptophysin (C), PSD-95 (D), or F-actin (E). Arrowheads in C indicate that the immunolabeling for DA (green) was closely adjacent to the staining for synaptophysin (red). Examples of sites for co-localization (yellow) of DA (green) with either PSD-95 (red) or F-actin (red) are indicated by arrowheads in D and E, respectively. Arrow in D indicates a rare instance in which a DA-positive dot was not positive for PSD-95. Similar results were obtained in three independent experiments. Scale bars: 2 µm in C, D, E and in insets.

• Supplemental Figure S2 -

  Fig. S2. DA-GFP induces a reorganization of F-actin via interaction with its actin-binding domain. Cells transfected with GFP (A₁), DA-GFP (B₁), GFP-DAΔABS (C₁), and ABS-GFP (D₁) were stained with Texas red-X phalloidin to assess their F-actin organization (A₂, B₂, C₂, and D₂). (A₃) Corresponds to the merge of panel A₁ and A₂. (B₃) Merge of panels B₁ and B₂. (C₃) Merge of panels C₁ and C₂. (D₃) Merge of panels D₁ and D₂. Blue colour reveals nuclear staining by Hoechst 33258. Similar results were obtained in three independent experiments. Scale bar: 10 µm.

• Supplemental Figure S3 -

  Fig. S3. DA-GFP stabilizes F-actin via interaction with its actin-binding domain. Effects of the microfilament-depolymerizing drug cytochalasin B (10 µg/ml, 10 minutes) in cells transfected with GFP (A₁), DA-GFP (B₁), GFP-DAΔABS (C₁), and ABS-GFP (D₁). F-actin organization in these cells was assessed with Texas red-X phalloidin (A₂, B₂, C₂ and D₂). (A₃) Merge of panels A₁ and A₂. (B₃) Merge of panels B₁ and B₂. (C₃) Merge of panels C₁ and C₂. (D₃) Merge of panels D₁ and D₂. Blue color reveals nuclear staining with Hoechst 33258. Scale bar: 10 µm.

• Supplemental Figure S4 -

  Fig. S4. Effects of lipofectamine treatment on mEPSCs charge transfer recorded in hippocampal pyramidal cells. The mEPSCs were recorded from (1) neurons untreated neither with lipofectamine nor with oligonucleotides (UT); (2) neurons treated with DA-sense oligonucleotides and lipofectamine untreated (S); (3) non-transfected neurons in GFP transfected cultures (NT-GFP); (4) non-transfected neurons in DA-GFP transfected cultures (NT-DA-GFP); (5) GFP transfected neurons (GFP). (A) Plot illustrates the geometrical means of mEPSCs charge transfer for each recorded cell. (B) Histogram showing the average charge transfer of mEPSCs recorded in differentially treated neurons. The mEPSCs charge transfer of lipofectamine-treated cells was significantly reduced (NT-GFP: 0.057±0.019, n=5; NT-DA-GFP: 0.074±0.049, n=3; GFP: 0.071±0.028 pC/s, n=5) compared with that of lipofectamine-untreated cells (UT: 0.315±0.078 pC/second, n=7; P<0.05; Kruskall-Wallis test with Mann-Whitney U post hoc test). Thus, the different experimental procedures explain the difference between control values of DA-overexpressing experiments and control values of DA-downregulation experiments. In addition, there is no difference for mEPSCs charge transfer values between the different controls (NT-GFP, NT-DA-GFP, and GFP; P=0.8, Kruskall-Wallis test) recorded in lipofectamine-treated cultures. Therefore, the use of GFP transfected neurons is the appropriate control for DA-GFP transfected cells. It has been previously shown that DA sense oligonucleotides treatment has no effect on the regulation of the expression of DA and β-actin (Takahashi et al., 2003 and 2006). This issue was reexamined in our experimental conditions and our data showed that indeed DA sense oligonucleotides treatment did not alter the expression of DA as well as β3-tubulin as compared to untreated cultures (Ctl, see Fig. 6A,B). In addition, the DA sense oligonucleotides treatment (S) did not change the mEPSCs charge transfer compared with untreated cultures (S: 0.314±0.091 vs UT: 0.315±0.078 pC/second, n&γτ;6; P=0.5; Mann-Whitney U test). Therefore, sense-treated neurons are the appropriate control cells for the analysis of neuronal activity of DA antisense oligonuclotides-treated cells. To make a statistical analysis of multiple data groups of mEPSCs charge transfer parameter, we used Kruskall-Wallis One-Way Analysis of Variance, a non-parametric test equivalent to ANOVA. To evaluate the statistical significance of the difference between different groups we used Mann-Whitney U test with post-hoc Bonferroni correction for multiple comparisons. **P<0.01, *P<0.05.