Data for QTL analysis and regression analysis

Three spread sheets showing data for the QTL analysis and the regression analysis condunted in this paper

Three-dimensional reconstruction of perforant path-GC synapses in the dentate gyrus.

<p>(A, B) Full field SEM images obtained through FIB/SEM show the cross-sections of dendrites (green) in the placebo- (A) and fluoxetine (B)-treated mice. Insets show images of dendritic spines (green) and connecting boutons obtained in other sections. The arrows (red) indicate PSD. (C, D) 3D-reconstructed dendritic segments in the middle molecular layer of the DG in the placebo- (C) and fluoxetine (D)-treated mice. Note the appearance of the large-sized spines and PSDs (red) in the fluoxetine-treated mice. The dendritic spines, which are shown in the insets of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0147307#pone.0147307.g001" target="_blank">Fig 1A and 1B</a>, are indicated with numbers. (E, F) Three-dimensional reconstructed presynaptic boutons are visualized at two synapses in the placebo- (E) and fluoxetine (F)-treated mice. The synaptic vesicles (orange) and mitochondria (purple) are shown inside the presynaptic boutons. Note that the large-sized spines are in contact with large-sized presynaptic boutons. Scale bars: 1 μm.</p

Characterization of presynaptic boutons connected to spines of different sizes.

<p>(A) Presynaptic boutons were classified by their connections to spines with different sizes. The spines were subdivided into four groups (Groups 1–4) based on the mean and SD values of the spine volume in placebo-treated mice. Typical images of SEM and 3D reconstructed spines (green) and connected boutons (light brown) are shown. Synaptic vesicles (orange) and mitochondria (purple) are shown inside the presynaptic boutons. Scale bars: 1 μm. (B) Volume of presynaptic boutons connected to spines classified as Groups 1–4. n = 6–10 presynaptic boutons in each group. No significant difference between placebo and fluoxetine with two-way ANOVA (drug effect, F_(1,44) = 0.0682, p < 0.794; group effect, F_(3,44) = 11.5, <i>p</i> < 0.0001; drug and group interaction, F_(3,44) = 0.230, <i>p</i> = 0.875). One-way ANOVA: placebo, F_(3,20) = 4.48, <i>p</i> = 0.0147; fluoxetine, F_(3,24) = 7.81, <i>p</i> = 0.0008; Bonferroni’s post-hoc test, *<i>p</i> < 0.05, compared with Group 1 in placebo, ^†<i>p</i> < 0.05 compared with Group 2 in placebo, **<i>p</i> < 0.01 compared with Group 1 in fluoxetine, ^††<i>p</i> < 0.01 compared with Group 2 in fluoxetine, ^§<i>p</i> < 0.05 compared with Group 3 in fluoxetine. (C) Correlation between volumes of mitochondria and presynaptic boutons in mice treated with placebo (r² = 0.79, <i>p</i> < 0.0001) or fluoxetine (r² = 0.84, <i>p</i> < 0.0001) with linear regression analysis. The symbol indicates the number of mitochondrion in each presynaptic bouton: ●, one; ○, two; ▲, three. The mitochondrial volume is the sum of the volume for all of the mitochondria in each bouton. (D) Correlation between the volumes of synaptic vesicles and presynaptic boutons in mice treated with placebo (r² = 0.91, <i>p</i> < 0.0001) or fluoxetine (r² = 0.72, <i>p</i> < 0.0001) with linear regression analysis.</p

Spine volume and density in the placebo- and fluoxetine-treated mice.

<p>(A) The scatter plot shows the spine volume in mice treated with placebo (n = 207 spines from 9 dendrites, 3 dendrites per each of 3 mice) or fluoxetine (n = 175 spines from 9 dendrites, 3 dendrites per each of 3 mice). (B, C) Mean values of spine volume for all spines (Mann-Whitney <i>U</i>-test: U = 15930, p = 0.0425) (B) and spine density (Mann-Whitney <i>U</i>-test: U = 28, p = 0.2973) (C) in the placebo- or fluoxetine-treated mice. *<i>p</i> < 0.05 compared with the placebo-treated mice.</p

Effect of chronic fluoxetine treatment on optical responses in the hippocampal DG.

<p>(A) Effect of chronic fluoxetine treatment on optical responses evoked by the stimulation of the perforant path inputs in hippocampal slices. The left-most panel shows the pseudocolor image of the slice preparation in which the optical recordings were made. A series of optical images of neuronal activity were recorded at 1.2-ms intervals from 0 to 10.8 ms after nerve stimulation. The signal intensity, expressed as fractional changes in optical absorbance relative to the background (%), was coded by the pseudocolor image. (B, C) The maximum propagation area of the optical signal above the background noise was analyzed 7.2–9.6 ms after stimulation, when the activation of the dendrites of the GCs via glutamatergic synaptic transmission was detected (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0147307#pone.0147307.s001" target="_blank">S1 Fig</a>). Typical images of the excitation area (B), traces of the optical responses at the boxed area (B) and the quantified excitation area (C) are shown in mice treated with placebo (n = 16 slices from 8 mice) and fluoxetine (n = 19 slices from 9 mice). *<i>p</i> < 0.001 compared with placebo-treated mice; Mann-Whitney <i>U</i>-test (U = 50, p = 0.0008). The preliminary data used for these figures are reported in a review article in Japanese [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0147307#pone.0147307.ref071" target="_blank">71</a>].</p

PSD volume and its correlation with spine volume.

<p>(A) Scatter plot showing the PSD volume in mice treated with placebo (n = 173 PSDs from 9 dendrites, 3 dendrites per each of 3 mice) or fluoxetine (n = 160 PSDs from 9 dendrites, 3 dendrites per each of 3 mice). (B) Mean values of the PSD volume in the placebo- and fluoxetine-treated mice. *<i>p</i> < 0.05 compared with placebo-treated mice; Mann-Whitney <i>U</i>-test (U = 11920, p = 0.0284). (C) Correlation between the PSD volume and spine volume in the placebo or fluoxetine-treated mice. The fitted lines for the mice treated with placebo (r² = 0.63, <i>p</i> < 0.0001) or fluoxetine (r² = 0.64, <i>p</i> < 0.0001) were obtained through a linear regression analysis.</p

ΔFosB binds to PSMC5 in vitro.

<p>A. Schematic of ΔFosB, ΔFosB-LZM in which the leucine zipper domain is mutated to obliterate ΔFosB heterodimerization with Jun proteins, and Δ2ΔFosB which lacks the first 78 amino acids of the ΔFosB N-terminus. B. Schematic of PSMC5, PSMC5-NT which comprises the first 151 amino acids of PSMC5, PSMC5-CT which lacks the first 235 amino acids of PSMC5, and PSMC5-ΔCC which lacks the coiled-coil domain (amino acids 28–68). The AAA domain corresponds to a motif, ATPases Associated with diverse cellular Activities, present in many ATPases. C. 2.4 μg of pcDNA3.1-ΔFosB (lanes 1–4) or ΔFosB-LZM (lane 5) was co-transfected with 2.4 μg of FLAG-tagged PSMC5 or various deletion mutants into Neuro2a cells. Two days after transfection, cells were lysed and subjected to immunoprecipitation with an anti-FLAG antibody and then Western blotted with anti-ΔFosB or anti-FLAG antibody. Note that ΔFosB, but not ΔFosB-LZM, binds robustly to PSMC5 or PSMC5-NT, but not PSMC5-CT or PSMC5-ΔCC. The data shown in the figure were replicated in triplicate in each of three separate experiments.</p

PSMC5 regulation of FosB/ΔFosB expression in Rat 1A cells.

<p>A. Rat 1A cells were transfected with 4 μg of PSMC5 or control DNA. PSMC5 overexpression had no effect on basal expression levels of FosB or ΔFosB protein as determined by Western blotting, but produced a small but significant increase in the induction of ΔFosB by serum stimulation (F(2,21) = 9.75, p = 0.001). B. Rat 1A cells were transfected with 5 pmol of either of two siRNAs or scrambled RNA (control). Both siRNAs effectively decreased PSMC5 protein levels compared to control conditions (siRNA #1, 23 ± 5% of control; siRNA #2, 18 ± 6%; p<0.05; n = 4). PSMC5 knockdown had no effect on basal levels of FosB or ΔFosB but attenulated the induction of both FosB and ΔFosB by serum stimulation (FosB: F(2,6) = 20.99, p = 0.002; ΔFosB: F(2,6) = 22.83, p = 0.002).</p

PSMC5 overexpression in NAc increases locomotor responses to cocaine.

<p>A. Representative HSV-mediated transgene expression in medial NAc. AC, anterior commissure. NAc core and shell subregions are noted on the figure. B. Representative higher magnifications (60x) of immunohistochemical staining of PSMC5 in NAc neurons after HSV-PSMC5 injection showing that the protein is predominantly nuclear as marked by DAPI staining. C. Mice received bilateral HSV injections into NAc followed by daily IP injections of subthreshold doses of cocaine (7.5 mg/kg). Locomotor responses are shown in response to the first and last of 3 daily doses of the drug. Overexpression of PSMC5 or PSMC5-K196M increases locomotor responses to repeated cocaine, an effect not seen with PSMC5-ΔCC. There was no significant effect of the transgenes on locomotor responses to initial cocaine doses. ANOVA F(3,125) = 4.163, *p<0.05 by Dunnett’s posthoc test.</p

Results of Yeast Two-Hybrid Screening with Δ2ΔFosB.

<p>*Although PSMC5 is categorized as a subunit of the 26S proteasome, the 26S proteasome is composed of 19S and 20S particles. PSMC5 is a subunit of the 19S particle, which contains the ATPase activity of the proteasome.</p><p>Results of Yeast Two-Hybrid Screening with Δ2ΔFosB.</p