A genomic approach to interneuron diversity and the emergence of an epileptic brain

The hippocampus is an archicortical region involved in functions including memorisation and spatial navigation. These operations depend on complex synaptic interactions involving both excitatory and inhibitory signaling between hippocampal neurones. Changes in the balance of excitatory and inhibitory systems may result in pathological conditions such as epilepsy. Understanding the properties of hippocampal cells and networks at multiple levels and in both physiological and pathological conditions is thus an important task for research on the healthy and diseased brain. Genomic tools provide access to an essential level of organisation, that of gene expression and regulation in hippocampal cells. They may serve as a marker to study how a cell population responds to a physiological stimulus or identify genes specific to a defined cell type. Genetic approaches are also used to examine how gene expression is changed during pathological conditions and may help identify novel processes or molecules thus providing new pharmacological targets. In the first part of my thesis, microarray analysis of gene expression was used to compare patterns of gene expression in a subfamily of Somatostatin containing hippocampal interneurons and principal glutamatergic excitatory cells. GABAergic interneurons constitute a heterogeneous group while pyramidal cells are probably a rather homogeneous population. Interneurone diversity is important for the function hippocampal networks, and this genomic analysis will help understand this diversity. I found significant differences in genes expressed in interneurones and pyramidal cell populations. Protein products of differentially expressed genes are mainly involved in transport and signalling, together with some proteins coding for neurotransmitter receptors and channels and a cluster of transcription factors. Although these data need to be confirmed with RT PCR and immunocytochemical experiments, further work is necessary to identify genes involved in the development of the distinct phenotypes, in the control of different physiological properties or for use as cell type markers. The second part of my thesis examined changes in gene expression during the establishment of an epileptic network after intra-hippocampal injection of kainic acid. Many genes were changed with different time courses and spatial localisation with respect to the injection site. The altered genes are often involved in immune and inflammation responses but also in cell death and growth processes. Some genes coding for proteins that control cellular excitability and neuronal communication were also changed. The major implication of inflammatory and immune processes is consistent with previous work on animal models of epilepsy or human epileptic tissue. I confirmed changes in some individual genes with qPCR analysis. My work suggests that kainate injection changes the expression of early response genes near the lesion at 6hrs, but also induces distinct alterations at distant sites in contralateral hippocampus. A maximum number of changed genes was identified during the latent phase at 15 days after kainate injection but before the emergence of spontaneous seizures. At 6 months, recurrent spontaneous seizures have emerged and changes in gene expression are limited to the area near the lesion. The progression of epilepsy in this animal model was confirmed with EEG and slice records and anatomical work was done to characterize the time course of cell death and fibre degeneration. Alterations in gene regulation correspond quite well to these electrical and anatomical data. Furthermore immuno-histochemical stains for specific proteins produced by differentially regulated genes revealed their expression by proliferating astrocyte precursors and by activated astroglial cells that were differentially localized in the two hippocampi. In conclusion multiple different processes are triggered with different spatial patterns and timing during the emergence of an epileptic network. However, during the expression of recurrent seizures, few genes are changed except at sites surrounding the sclerotic lesion.