Fig 1.
Kainate induced seizures and neuronal damage.
A) Percentage of animals showing seizures after kainite administration. The intensity of seizure was scored according to the 6-grade modified Racine’s scale (0-lack of seizures—5- fully developed tonic-clonic seizures with loss of posture). B) Fluoro-jade B staining (depicted in green) showing neuronal damage in the CA3 region, but not the DG region of the hippocampus of kainite treated animals. Scale Bar: 100 μm.
Table 1.
Estimation of the clinical and morphological traits in the kainate model of TLE.
Fig 2.
Correlation between the intensity of sprouting and the percentage of the nuclei with Bdnf alleles at the nuclear periphery.
A) Mossy fiber sprouting was verified by immunofluorescent staining for synaptoporin in the molecular layer of DG region of the hippocampus. Representative pictures of different levels of sprouting of animals at 4 weeks after administration of kainate are shown. B) Percentage of animals showing different levels of synaptoporin staining intensity scored in 5-grade scale (0-lack of sprouting- 4 very strong sprouting). C) Correlation between the levels of sprouting in the DG, 4 weeks from the administration of kainate, measured in 5- grade scale, and percentage of the nuclei with Bdnf alleles localized at the nuclear periphery. Scale Bar: 100 μm.
Fig 3.
The causal relationship between Bdnf transcriptional activity and Bdnf’s allele repositioning.
(A) The graph shows the expression of Bdnf normalized to control in the hippocampal neurons incubated for 2 hours with DMSO vehicle (CTRL, blue bar) or picrotoxin, forskolin, and rolipram (cLTP, orange bar), incubated for 2 hours with Actinomycin D and 2 hours with DMSO (ActD, green bar) or picrotoxin, forskolin, and rolipram (ActD+cLTP, red bar). Kruskal-Wallis group comparison: p<0.01, and the One-Sample T-test or Welch Two Sample t-test for pairwise comparison: * p<0.05, **p<0.01, *** p<0.001; error bars indicate standard error of the mean for 3 independent experiments (B) Representative picture of the nuclei of hippocampal neurons treated as described above. Hoechst 3342 staining for chromatin is shown in greyscale and segmentation of FISH signals for the Bdnf gene are shown in magenta and cyan. (C) Percentages of Bdnf alleles localized < 350 nm to the nuclear surface are shown (Chi-square test, all groups, p<0.001; Fisher’s exact tests * p<0.05, *** p<0.001, error bars indicate standard deviation of the binomial distribution). (D) Quantitative analysis of the intracellular positions of Bdnf alleles in the nuclei of hippocampal neurons treated and color-coded as in A. The minimal distance between the respective alleles and nucleus surface is presented in the normalized histogram.
Fig 4.
The inhibition of histone deacetylases induces Bdnf transcription and repositioning independently from the neuronal stimulation.
(A) The graph shows the expression of Bdnf normalized to control in the hippocampal neurons incubated for 2 hours with DMSO vehicle (CTRL, blue bar) or picrotoxin, forskolin, and rolipram (cLTP, orange bar), incubated for 12 hours with TSA and 2 hours with DMSO (TSA, cyan bar) or picrotoxin, forskolin, and rolipram (TSA+cLTP, magenta bar). Kruskal-Wallis group comparison p<0.01, and the One-Sample T-test or Welch Two Sample t-test for pairwise comparison: * p<0.05, **p<0.01, *** p<0.001; error bars indicate standard error of the mean for 5 independent experiments (B) Representative pictures of the nuclei of hippocampal neurons treated as described above. Hoechst 3342 staining for chromatin is shown in greyscale and segmentation of FISH signals for the Bdnf gene are shown in magenta and cyan. (C) Percentages of nuclei with the minimum distance between the respective alleles and nucleus surface < 350 nm are shown (Chi-square test, all groups, p<0.01; Fisher’s exact tests * p<0.05, *** p<0.001, error bars indicate standard deviation of the binomial distribution). (D) Quantitative analysis of the intracellular positions of Bdnf alleles in the nuclei of hippocampal neurons treated and color-coded as in A. The minimal distance between the respective alleles and nucleus surface is presented in the normalized histogram.
Fig 5.
The inhibition of HDAC1/2 is not sufficient to induce Bdnf transcription and repositioning.
(A) The graph shows the expression of Bdnf relative to Gapdh (normalized to control Bdnf level) in the hippocampal neurons incubated for 2 hours with DMSO vehicle (CTRL, blue bar) or picrotoxin, forskolin, and rolipram (cLTP, orange bar), incubated for 2 hours with 250 nM romidepsin and 2 hours with DMSO (romidepsin, light blue bar) or picrotoxin, forskolin, and rolipram (romidepsin+cLTP, violet bar). Kruskal-Wallis test for group comparison: p<0.01, One-Sample t-test or Welch two sample t-test for pairwise comparison: * p<0.05, *** p<0.001; error bars indicate standard error of the mean for 3 independent experiments (B) Representative picture of the nuclei of hippocampal neurons treated as described above. Hoechst 3342 staining for chromatin is shown in greyscale and segmentation of FISH signals for the Bdnf gene are shown in magenta and cyan. (C) Percentages of nuclei with the minimum distance between the respective alleles and nucleus surface < 350 nm are shown (Chi-square test, all groups, p<0.0001; Fisher’s exact tests: ** p<0.01, *** p<0.001, error bars indicate standard deviation of the binomial distribution). (D) Quantitative analysis of the intracellular positions of Bdnf alleles in the nuclei of hippocampal neurons treated and color-coded as in A. The minimal distance between the respective alleles and nucleus surface is presented in the normalized histogram.