Hippocampal spine changes across the sleep–wake cycle: corticosterone and kinases

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   Figure 1

   Diurnal change of the spine density in hippocampal CA1 pyramidal neurons. Spines were analyzed along the secondary dendrites of CA1 pyramidal neurons in the stratum radiatum every 3 h. (A) Representative images of confocal micrographs; the spines along dendrite at Zeitgeber time 4 (ZT4, 1200 h), ZT10 (1800 h), ZT13 (2100 h), and ZT16 (2400 h). Maximal intensity projections onto XY plane from z-series confocal micrographs (MAX-XY), images analyzed by Spiso-3D (Spiso), and three-dimensional model illustrations (model) are shown together. Bar, 5 μm. (B) Diurnal change of the total spine density in the hippocampus. Vertical axis represents the average number of spines per 1 μm of dendrite. Data are represented as mean±s.e.m. (C) Histogram of spine head diameters at ZT4 (closed white circle), ZT10 (closed blue circle), ZT13 (closed black circle), and ZT16 (closed green circle). (D) Density of three subtypes of spines at ZT1 (yellow column), ZT4 (white column), ZT7 (red column), ZT10 (blue column), ZT13 (black column), ZT16 (green column), ZT19 (orange column), and ZT22 (purple column). From left to right, small-head spines (small), middle-head spines (middle), and large-head spines (large) type. Data are represented as mean±s.e.m. The statistical significance was examined using one-way ANOVA followed by the Tukey–Kramer post hoc multiple comparisons test. The significance yielded: **P<0.01, *P<0.05 vs ‘ZT10’. For each period of time, we investigated three to four rats, six to eight slices, 30–40 neurons, 60–80 dendrites, and ∼6000–8000 spines. A full colour version of this figure is available at http://dx.doi.org/10.1530/JOE-15-0078.
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   Figure 2

   Effect of adrenalectomy (ADX) on the diurnal change of the spine density. Endogenous CORT was depleted by ADX, and spine density was analyzed at ZT4 (ZT4_ADX) and ZT13 (ZT13_ADX). (A) Representative images of confocal micrographs; the spines along the dendrite of ADX rats at ZT13. Maximal intensity projections onto XY plane from z-series confocal micrographs (MAX-XY), images analyzed by Spiso-3D (Spiso), and three-dimensional model illustrations (model) are shown together. Bar, 5 μm. (B) Effect of ADX on the total spine density. Vertical axis represents the average number of spines per 1 μm of dendrite. Data are represented as mean±s.e.m. (C) Histogram of spine head diameters at ZT4 (closed white circle) and ZT13 (closed black circle) and of ADX rats at ZT4 (closed red circle) and ZT13 (closed blue circle). (D) Density of three subtypes of spines at ZT4 (white column) and ZT13 (black column) and in ADX rats at ZT4 (red column) and ZT13 (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±s.e.m. The statistical significance yielded: **P<0.01 and *P<0.05. For each condition, we investigated three to four rats, six to eight slices, 30–40 neurons, 60–80 dendrites, and ∼6000–8000 spines. A full colour version of this figure is available at http://dx.doi.org/10.1530/JOE-15-0078.
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   Figure 3

   Effect of the administration of CORT on ADX rats. Exogenous CORT was administered subcutaneously (1 mg/kg body weight) on ADX rats at ZT11. Spine density was analyzed at ZT13 (ZT13_ADX+CORT). (A) Representative images of confocal micrographs; the spines along the dendrite of ADX rats with CORT administration. Maximal intensity projections onto XY plane from z-series confocal micrographs (MAX-XY), images analyzed by Spiso-3D (Spiso), and three-dimensional model illustrations (model) are shown together. Bar, 5 μm. (B) Effect of the administration of CORT on the total spine density. Vertical axis represents the average number of spines per 1 μm of dendrite. Data are represented as mean±s.e.m. (C) Histogram of spine head diameters at ZT4 (closed white circle) and ZT13 (closed black circle), in ADX rats at ZT13 (closed blue circle), and in CORT-administered ADX rats (closed green circle). (D) Density of three subtypes of spines at ZT4 (white column) and ZT13 (black column), in ADX rats at ZT13 (blue column), and in CORT-administered ADX rats (green column). From left to right, small-head spines (small), middle-head spines (middle), and large-head spines (large) type. Data are represented as mean±s.e.m. The statistical significance yielded: **P<0.01, *P<0.05 vs ‘ZT13’ and ‘ZT13_ADX’. For each condition, we investigated three to four rats, six to eight slices, 30–40 neurons, 60–80 dendrites, and ∼6000–8000 spines. A full colour version of this figure is available at http://dx.doi.org/10.1530/JOE-15-0078.
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   Figure 4

   Effect from the inhibition of CORT synthesis on the diurnal change of the spine density. The synthesis of CORT at the adrenal gland was inhibited by metyrapone. Vehicle (sesame oil (ZT13+sesame)) or 50 mg/kg body weight metyrapone was administered subcutaneously at ZT9 and spine density was analyzed at ZT13 (ZT13+metyra). (A) Representative images of confocal micrographs; the spines along dendrite of metyrapone administered group. Maximal intensity projections onto XY plane from z-series confocal micrographs (MAX-XY), images analyzed by Spiso-3D (Spiso), and three-dimensional model illustrations (model) are shown together. Bar, 5 μm. (B) Effect of metyrapone on the total spine density. Vertical axis represents the average number of spines per 1 μm of dendrite. Data are represented as mean±s.e.m. (C) Histogram of spine head diameters at ZT4 (closed white circle) and ZT13 (closed black circle), in metyrapone administered group (closed red circle), and in vehicle administered group (closed blue circle). (D) Density of three subtypes of spines at ZT4 (white column) and ZT13 (black column), in metyrapone administered group (red column), and in vehicle administered group (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±s.e.m. The statistical significance yielded: **P<0.01 vs ‘ZT13’. For each condition, we investigated three to four rats, six to eight slices, 30–40 neurons, 60–80 dendrites, and ∼6000–8000 spines. A full colour version of this figure is available at http://dx.doi.org/10.1530/JOE-15-0078.
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   Figure 5

   Time dependency of CORT effects on the spine density in isolated hippocampal slices. Spines in isolated hippocampal slices were analyzed along the secondary dendrites of CA1 pyramidal neurons in the stratum radiatum. 30 nM CORT was treated on acute hippocampal slices for 1 h (CORT_1 h), 2 h (CORT_2 h), and 3 h (CORT_3 h) in ACSF. (A) Representative images of confocal micrographs; the spines along dendrite without drugs (control) and with 30 nM of CORT treatment for 1 h. Maximal intensity projections onto XY plane from z-series confocal micrographs (MAX-XY), images analyzed by Spiso-3D (Spiso), and three-dimensional model illustrations (model) are shown together. Bar, 5 μm. (B) Time dependency of CORT effects on the total spine density. As a control, no treatment with CORT for 1 h (control (1 h)) is shown. Vertical axis represents the average number of spines per 1 μm of dendrite. Data are represented as mean±s.e.m. (C) Histogram of spine head diameters after a 1 h treatment in ACSF without drugs (closed white circle) and with 30 nM CORT for 1 h (closed black circle), 2 h (closed red circle), and 3 h (closed blue circle). (D) Density of three subtypes of spines after a 1 h treatment in ACSF without drugs (white column) and with 30 nM CORT for 1 h (black column), 2 h (red column), and 3 h (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±s.e.m. The statistical significance yielded: **P<0.01 and *P<0.05. For each condition, we investigated three to four rats, six to eight slices, 30–40 neurons, 60–80 dendrites, and ∼3000–4000 spines. A full colour version of this figure is available at http://dx.doi.org/10.1530/JOE-15-0078.
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   Figure 6

   Effects by the blockade of receptors and inhibition of gene expressions on CORT-induced spinogenesis. (A) Representative images of confocal micrographs; the spines along dendrite with 30 nM CORT and 10 μM RU486 (blocker of glucocorticoid receptor (GR)) (CORT+RU) for 1 h. Maximal intensity projections onto XY plane from z-series confocal micrographs (MAX-XY), images analyzed by Spiso-3D (Spiso) and three-dimensional model illustrations (model) are shown together. Bar, 5 μm. (B) Effects from the blocker of GR and mineralocorticoid receptor (MR) on the CORT-induced spinogenesis. A 1 h treatment in ACSF without drugs (control), with 30 nM CORT, with 30 nM CORT and 10 μM RU486 (CORT+RU), and with 30 nM CORT and 10 μM spironolactone (blocker of MR) (CORT+Spiro). Vertical axis represents the average number of spines per 1 μm of dendrite. Data are represented as mean±s.e.m. (C) Effects by the inhibition in mRNA and protein synthesis on the CORT-induced spinogenesis. A 1 h treatment in ACSF without drugs (control), with 30 nM CORT, with 30 nM CORT and 4 μM actinomycin D (translation inhibitor) (CORT+ActD), and with 30 nM CORT and 20 μM cycloheximide (transcription inhibitor) (CORT+CHX). Vertical axis represents the average number of spines per 1 μm of dendrite. Data are represented as mean±s.e.m. The statistical significance yielded: **P<0.01 vs ‘CORT’. For each drug treatment, we investigated three to four rats, six to eight slices, 30–40 neurons, 60–80 dendrites, and ∼3000–4000 spines. A full colour version of this figure is available at http://dx.doi.org/10.1530/JOE-15-0078.
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   Figure 7

   Suppression effects by kinase inhibitors on CORT-induced spinogenesis. Kinase inhibitors were co-treated with 30 nM CORT on isolated hippocampal slices. A 1 h treatment in ACSF without drugs (control), with 30 nM CORT (CORT), with 30 nM CORT and 10 μM H89 (PKA inhibitor) (CORT+H89), with 30 nM CORT and 10 μM chelerythrine (PKC inhibitor) (CORT+Chel), with 30 nM CORT and 25 μM U0126 (ERK inhibitor) (CORT+U), with 30 nM CORT and 10 μM LIMK inhibitor (CORT+LIMKi), and with 30 nM CORT and 10 μM SP600125 (JNK inhibitor) (CORT+SP). (A) Representative images of confocal micrographs; the spines along dendrite with CORT and H89, with CORT and chelerythrine, with CORT and U0126, with CORT and LIMK inhibitor. Maximal intensity projections onto XY plane from z-series confocal micrographs (MAX-XY), images analyzed by Spiso-3D (Spiso), and three-dimensional model illustrations (model) are shown together. Bar, 5 μm. (B) Effects by the co-treatment of kinase inhibitors on the total spine density. Vertical axis represents the average number of spines per 1 μm of dendrite. Data are represented as mean±s.e.m. (C) Histogram of spine head diameters after a 1 h treatment in ACSF without drugs (closed white circle), with CORT (closed black circle), with CORT and H89 (closed red circle), with CORT and chelerythrine (closed blue circle), with CORT and U0126 (closed green circle), with CORT and LIMK inhibitor (closed orange circle), and with CORT and SP600125 (closed purple circle). (D) Density of three subtypes of spines after a 1 h treatment in ACSF without drugs (white column), with CORT (black column), with CORT and H89 (red column), with CORT and chelerythrine (blue column), with CORT and U0126 (green column), with CORT and LIMK inhibitor (orange column), and with CORT and SP600125 (purple column). From left to right, small-head spines (small), middle-head spines (middle), and large-head spines (large) type. Data are represented as mean±s.e.m. The statistical significance yielded: **P<0.01, *P<0.05 vs ‘CORT’. For each drug treatment, we investigated three to four rats, six to eight slices, 30–40 neurons, 60–80 dendrites, and ∼3000–4000 spines. A full colour version of this figure is available at http://dx.doi.org/10.1530/JOE-15-0078.
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   Figure 8

   Schematic illustration of CORT-driven multiple kinase pathways. Upon binding of CORT, GR induces the sequential activation of PKA, PKC, ERK MAPK, and LIMK. LIMK phosphorylates cofilin, resulting in actin reorganization and subsequent spinogenesis, whereas ERK/MAPK phosphorylates cortactin. A full colour version of this figure is available at http://dx.doi.org/10.1530/JOE-15-0078.

• © 2015 Society for Endocrinology