Supplementary Figure

• Supplementary Figure 1 - Model illustration for preparing isolated hippocampal slices The level of CORT in slices is determined by mass-spectrometric analysis (Hojo et al. 2011). (A) Freshly isolated hippocampus which contains approx. 150–500 nM CORT. (B) Preparation of hippocampal “fresh” slices. (C) For preparation of “acute slices”, slices are incubated for recovery in steroid-free ACSF for 2h, resulting in depletion of steroids. (D) Acute hippocampal slices contain approx. 2 nM CORT. (PDF 31 KB)
• Supplementary Figure 2 - Calibration curves for LC-MS/MS using standard CORT dissolved in ethanol The calibration curve for CORT. Linearity is observed between 2 pg/mL and 4000 pg/mL. Horizontal axis indicates the concentration of added standard steroid. Vertical axis indicates the relative intensity obtained from the chromatogram. (PDF 31 KB)
• Supplementary Figure 3 - Diurnal change in the level of CORT in the cerebrospinal fluid (CSF) from the cisterna magna in freely moving rats Rats with a microdialysis probe are maintained in the 12 h light/12 h dark cycle. Samples are collected every hour and 2 or 3 samples are combined and averaged. Data are represented as mean ± SEM. Three independent experiments with different animals were performed for each of these analyses, showing good reproducibility. [Data are modified and corrected from Higo et al. (Higo et al. 2011)] (PDF 31 KB)
• Supplementary Figure 4 - Representative dendritic spine images showing the effects of ADX and several blockers (Spirono, ActD, CHX and SP) on spinogenesis (A) Representative images of confocal micrographs; the spines along dendrite in ADX rats at ZT4 and in sesame oil administrated group. (B) Representative images of confocal micrographs; spines along dendrite with 30 nM CORT and 10 μM spironolactone (CORT + Spiro), with 30 nM CORT and 4 μM actinomycin D (CORT + ActD), with 30 nM CORT and 20 μM cycloheximide (CORT + CHX), and with 30 nM CORT and 10 μM SP600125 (CORT + SP) for 1 h. Maximal intensity projections onto XY plane from z-series confocal micrographs (MAX-XY), images analyzed by Spiso-3D (Spiso) and 3 dimensional model illustrations (Model) are shown together. Bar, 5 μm.(PDF 31 KB)
• Supplementary Figure 5 - Mass-spectrometric analysis of hippocampal corticosteroids after CORT administration on ADX rats LC-MS/MS ion chromatograms of CORT. (A) The chromatograms of the fragmented ions of CORT (m/z = 121) from the hippocampus of ADX rats, two hours after the CORT administration (s.c., 1 mg/kg body weight). Shaded portions indicate the intensity of fragmented ions. (B) The chromatograms of the fragmented ions in the standard CORT. The vertical axis represents the intensity of the fragmented ion. The horizontal axis indicates the retention time of the fragmented ion, t = 3.19 min. The time of sample injection to LC system was defined as t = 0 min. Note that pre-purification step using normal phase HPLC before injection to LC system is very important to achieve high precision and good reproducibility of LC-MS/MS determination in order to avoid contamination of other steroids and fats. Steroids are further separated with reversed phase LC-column. In the multiple reaction monitoring mode, the instrument monitored the m/z transition. (PDF 31 KB)
• Supplementary Figure 6 - Time dependency of the spine density in the control condition The isolated hippocampal slices were incubated without drugs (control condition) for 1 h, 2 h, and 5 h. Vertical axis is the average number of spines per 1 μm of dendrite. Data are represented as mean ± SEM. For each condition, we investigated 3–4 rats, 6–8 slices, 30–40 neurons, 60–80 dendrites, and approx. 3000–4000 spines. (PDF 31 KB)
• Supplementary Figure 7 - Effects from blockers and inhibitors alone on the spine density (A) A 1 h treatment in ACSF without drugs (Control), with 10 μM RU486 (RU), and with 10 μM spironolactone (Spiro). (B) A 1 h treatment in ACSF without drugs (Control), with 20 μM cycloheximide (CHX), and with 4 μM actinomycin D (ActD). (C) A 1 h treatment in ACSF without drugs (Control), with 10 μM H89, with 10 μM chelerythrine (Chel), with 25 μM U0126, with 10 μM LIMKi, and with 10 μM SP600125 (SP). Vertical axis represents the average number of spines per 1 μm of dendrite. Data are represented as mean ± SEM. For each drug treatment, we investigated 3–4 rats, 6–8 slices, 30–40 neurons, 60–80 dendrites, and approx. 3000–4000 spines. (PDF 31 KB)
• Supplementary Figure 8 - Head diameter distribution for effects by the blockade of receptors and gene transcription on CORT-induced spinogenesis (A) Histogram of spine head diameters after a 1 h treatment in ACSF without drugs (closed white circle), with 30 nM CORT (closed black circle), with 30 nM CORT and 10 μM RU486 (CORT + RU) (closed red circle), and with 30 nM CORT and 10 μM spironolactone (CORT + Spiro) (closed blue circle). (B) Density of three subtypes of spines after a 1 h treatment in ACSF without drugs (white column), with 30 nM CORT (black column), with 30 nM CORT and 10 μM RU486 (red column), and with 30 nM CORT and 10 μM spironolactone (blue column). From left to right, small-head spines (small), middle-head spines (middle), and large-head spines (large) type. Abbreviations are same as in (A). Data are represented as mean ± SEM. (C) Histogram of spine head diameters after a 1 h treatment in ACSF without drugs (closed white circle), with 30 nM CORT (closed black circle), with 30 nM CORT and 4 μM actinomycin D (CORT + ActD) (closed red circle), and with 30 nM CORT and 20 μM cycloheximide (CORT + CHX) (closed blue circle). (D) Density of three subtypes of spines after a 1 h treatment in ACSF without drugs (white column), with 30 nM CORT (black column), with 30 nM CORT and 4 μM actinomycin D (CORT + ActD) (red column), and with 30 nM CORT and 20 μM cycloheximide (CORT + CHX) (blue column). From left to right, small-head spines (small), middle-head spines (middle), and large-head spines (large) type. Data are represented as mean ± SEM. The statistical significance yielded *P < 0.05 vs. “CORT”. For each drug treatment, we investigated 3–4 rats, 6–8 slices, 30–40 neurons, 60–80 dendrites, and approx. 3000–4000 spines. (PDF 31 KB)
• Supplementary Figure 9 - Dose dependency of CORT treatments on the spine density in isolated hippocampus A 1 h treatment in ACSF without drugs (Control, 2 nM CORT), with 10 nM CORT, and with 30 nM CORT. Data are represented as mean ± SEM. For each condition, we investigated 3–4 rats, 6–8 slices, 30–40 neurons, 60–80 dendrites, and approx. 3000–4000 spines. (PDF 31 KB)