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.
Highlights
Cognitive processes such as learning and memory originate from plastic modifications in the central nervous system CNS: these plastic changes affect the structure and the functions of neurons and of synapses and lead to experience-dependent alterations in neural network wiring and behavior
We decided to start the analysis with a preliminary, microarray-based, assessment of the response of rat organotypic hippocampal cultures to a 20 mM gabazine treatment (GabT): the purpose of this step was to obtain a complete profile of the tissue reaction to a prolonged GABA-A receptor blockade, which is strictly associated with a sudden and powerful increase in the tissue synaptic activity and in the intensity of calcium dynamics [6][23][24]
The present article identifies three different dynamical patterns in the early-phase (10–90 min) of the transcriptional response induced by GabT of organotypic hippocampal cultures and provides novel information about the role of Serum Response Factor
Summary
Cognitive processes such as learning and memory originate from plastic modifications in the central nervous system CNS: these plastic changes affect the structure and the functions of neurons and of synapses and lead to experience-dependent alterations in neural network wiring and behavior. The modulation of gene transcription has proven to be playing a key role in neuroplasticity: increased synaptic activity leads to calcium influx into the post-synaptic spines, dendrites and soma, which activates calcium dependent signaling pathways that in turn regulate transcription factors within the nucleus [4][5][6]. The expression level of many other plasticity-related genes is governed by sophisticated controls of dynamics [13]: this result is often achieved thanks to the interplay of a large number of transcription factors and is often related to signaling changes which are triggered within a time-scale of minutes [14][15][16]
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