Early phase of plasticity-related gene regulation and SRF dependent transcription in the hippocampus

PLoS One. 2013 Jul 23;8(7):e68078. doi: 10.1371/journal.pone.0068078. Print 2013.

Abstract

Hippocampal organotypic cultures are a highly reliable in vitro model for studying neuroplasticity: in this paper, we analyze the early phase of the transcriptional response induced by a 20 µM gabazine treatment (GabT), a GABA-Ar antagonist, by using Affymetrix oligonucleotide microarray, RT-PCR based time-course and chromatin-immuno-precipitation. The transcriptome profiling revealed that the pool of genes up-regulated by GabT, besides being strongly related to the regulation of growth and synaptic transmission, is also endowed with neuro-protective and pro-survival properties. By using RT-PCR, we quantified a time-course of the transient expression for 33 of the highest up-regulated genes, with an average sampling rate of 10 minutes and covering the time interval [10∶90] minutes. The cluster analysis of the time-course disclosed the existence of three different dynamical patterns, one of which proved, in a statistical analysis based on results from previous works, to be significantly related with SRF-dependent regulation (p-value<0.05). The chromatin immunoprecipitation (chip) assay confirmed the rich presence of working CArG boxes in the genes belonging to the latter dynamical pattern and therefore validated the statistical analysis. Furthermore, an in silico analysis of the promoters revealed the presence of additional conserved CArG boxes upstream of the genes Nr4a1 and Rgs2. The chip assay confirmed a significant SRF signal in the Nr4a1 CArG box but not in the Rgs2 CArG box.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Binding Sites
  • Brain-Derived Neurotrophic Factor / genetics
  • Brain-Derived Neurotrophic Factor / metabolism
  • Chromatin Immunoprecipitation
  • Cluster Analysis
  • Gene Expression Regulation* / drug effects
  • Gene Ontology
  • Hippocampus / drug effects
  • Hippocampus / metabolism*
  • Neuronal Plasticity / genetics*
  • Oligonucleotide Array Sequence Analysis
  • Pyridazines / pharmacology
  • RNA, Messenger / genetics
  • RNA, Messenger / metabolism
  • Rats
  • Rats, Wistar
  • Reproducibility of Results
  • Reverse Transcriptase Polymerase Chain Reaction
  • Serum Response Factor / metabolism*
  • Time Factors
  • Transcription, Genetic* / drug effects
  • Up-Regulation / drug effects
  • Up-Regulation / genetics

Substances

  • Brain-Derived Neurotrophic Factor
  • Pyridazines
  • RNA, Messenger
  • Serum Response Factor
  • gabazine

Grants and funding

The authors acknowledge the financial support of the SI-CODE project of the Future and Emerging Technologies (FET) programme within the Seventh Framework Programme for Research of the European Commission, under FET-Open grant number: FP7-284553.