Increased Histone H3 Phosphorylation in Neurons in Specific Brain Structures after Induction of Status Epilepticus in Mice

<div><p>Status epilepticus (SE) induces pathological and morphological changes in the brain. Recently, it has become clear that excessive neuronal excitation, stress and drug abuse induce chromatin remodeling in neurons, thereby altering gene expression. Chromatin remodeling is a key mechanism of epigenetic gene regulation. Histone H3 phosphorylation is frequently used as a marker of chromatin remodeling and is closely related to the upregulation of mRNA transcription. In the present study, we analyzed H3 phosphorylation levels <i>in vivo</i> using immunohistochemistry in the brains of mice with pilocarpine-induced SE. A substantial increase in H3 phosphorylation was detected in neurons in specific brain structures. Increased H3 phosphorylation was dependent on neuronal excitation. In particular, a robust upregulation of H3 phosphorylation was detected in the caudate putamen, and there was a gradient of phosphorylated H3⁺ (PH3⁺) neurons along the medio-lateral axis. After unilateral ablation of dopaminergic neurons in the substantia nigra by injection of 6-hydroxydopamine, the distribution of PH3⁺ neurons changed in the caudate putamen. Moreover, our histological analysis suggested that, in addition to the well-known MSK1 (mitogen and stress-activated kinase)/H3 phosphorylation/c-fos pathway, other signaling pathways were also activated. Together, our findings suggest that a number of genes involved in the pathology of epileptogenesis are upregulated in PH3⁺ brain regions, and that H3 phosphorylation is a suitable indicator of strong neuronal excitation.</p> </div

H3 phosphorylation level is also upregulated in specific brain structures of the kainic acid (KA)-induced SE brain.

<p>(A) H3 phosphorylation is greatly increased in the ipsilateral side in the KA-induced SE brain. (B) c-fos expression is also greatly upregulated in the ipsilateral side. Note that the distribution of c-fos⁺ neurons is much broader than that of PH3⁺ neurons, especially in the cerebral cortex. Stacked epifluorescence microscopy images are shown. Scale bar = 600 μm. (C) Densities of PH3⁺ neurons are significantly higher in the ipsilateral side compared with the contralateral side. Four animals were analyzed (<i>n</i> = 4). Two-tailed Welch’s <i>t</i>-test. *<i>p</i> < 0.05; **<i>p</i> < 0.01. White bars, ipsilateral side; gray bars, contralateral side.</p

Dopaminergic input from the substantia nigra affects H3 phosphorylation in the CPu of the pilocarpine-induced SE brain.

<p>Unilaterally 6-hydroxydopamine (6-OHDA)-injected mice were subjected to SE induction. Double immunostaining with anti-tyrosine hydroxylase (TH) and anti-PH3 antibodies was performed on the vehicle-administered control brain section (A, C and E) and the pilocarpine-induced SE brain section (B, D, F and G). TH immunoreactivity in the CPu and Acb is reduced significantly in the 6-OHDA injection side (A and B). H3 phosphorylation is significantly increased in the SE brain, especially in the ipsilateral side (D, F and G). Stacked epifluorescence microscopy images are shown. High magnification images of the boxed areas are shown in each image. (H) Density of PH3⁺ neurons in the CPu is significantly higher in the ipsilateral side than in the contralateral side of the pilocarpine-induced SE brain. White bar, ipsilateral side; gray bar, contralateral side. (I) Density gradient of PH3⁺ neuron in the CPu is lost in the 6-OHDA-injected side. White bar, medial part of the CPu; gray bar, lateral part of the CPu. Five animals were analyzed (<i>n</i> = 5). Two-tailed Welch’s <i>t</i>-test. *<i>p</i> < 0.05. Scale bar = 600 μm for A-D and 150 μm for E-G.</p

Identification of PH3⁺ cells in the CPu of the SE brain at 1 h.

<p>Identity of PH3⁺ cells (red) was determined by double immunostaining with cell type specific markers (green). Virtually all the PH3⁺ cells are NeuN⁺ mature neurons (A), not S100β⁺ astrocytes (B), Olig2⁺ oligodendrocytes/oligodendrocyte precursors (C) or Iba1⁺ microglia (D). Arrows indicate PH3⁺/NeuN⁺ (double-labeled) cells and arrowheads indicate PH3⁺ (single-labeled) cells. Single optical confocal microscopy images are shown. Scale bar = 50 μm.</p

MSK1 is phosphorylated in the pilocarpine-induced SE brain at 1 h.

<p>Stacked epifluorescence microscopy images of the anterior (A-E) and middle (F-H) part of the brain of the control (A and C) and SE 1 h group (B and D-H) are shown. High magnification images of the Acb (C and D), Cg (E), BNST (G), ventral limb of the CPu (arrowheads, G), and the somatosensory cortex (H) are shown. (A and C) In the control brain, there are virtually no phosphorylated MSK1⁺ (pMSK1⁺) cells. The distribution of pMSK1⁺ cells is similar to that of PH3⁺ neurons in the SE brain at 1 h (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077710#pone-0077710-g003" target="_blank">Figure 3</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077710#pone.0077710.s005" target="_blank">Figure S5</a>). Note that there are fewer pMSK1⁺ cells in the CPu (G). Cg: cingulate cortex. Scale bar = 600 μm for A, B and F, and 150 μm for C-E and G-H.</p

Distribution of PH3⁺ cells in the SE brain at 1 h.

<p>Anterior (A), middle (B) and posterior (C) parts are shown (see Materials and Methods). Stacked epifluorescence microscopy images are shown. Significant signals are detected in the CPu (A-C), nucleus accumbens (A), bed nucleus of the stria terminalis (B), interstitial nucleus of the posterior limb of the anterior commissure (B and C), dorsal endopiriform nucleus (A-C), piriform cortex (C), central amygdala (C), medial tuberal nucleus (D), ventromedial hypothalamic nucleus, ventrolateral part (D) and dentate gyrus of the hippocampus (E). (F) The CPu was divided into three parts with vertical lines (dotted lines in A-C; see Materials and Methods for detail), and the densities of PH3⁺ cells were compared between the medial and the lateral regions. Three animals were analyzed (<i>n</i> = 3). Two-tailed Welch’s <i>t</i>-test. **<i>p</i> < 0.01. White bars, medial region of CPu; gray bars, lateral regions of CPu; n.s., not significant. 3V: third ventricle; ac: anterior commissure; Acb: nucleus accumbens; BNST: bed nucleus of the stria terminalis; CeA: central amygdala; DEn: dorsal endopiriform nucleus; dG: dentate gyrus of the hippocampus; IPAC: interstitial nucleus of the posterior limb of the anterior commissure; LV: lateral ventricle; MTu: medial tuberal nucleus; Pir: piriform cortex; Tu: olfactory tubercle; VMHVL: ventromedial hypothalamic nucleus, ventrolateral part. Scale bar = 600 μm for A-D and 200 μm for E.</p