Transcriptome analysis of laser-microdissected rat brain regions relevant to cognition and psychiatric disorders

Genetics, Neurosciences, Mental health

Ferra, Antonio

University:University of Glasgow

关键词: Genetics, Neurosciences, Mental health;   

Others  :  http://theses.gla.ac.uk/70966/1/10390531.pdf

来源: University of Glasgow

【 摘 要 】

Schizophrenia is a severe psychiatric disorder associated with hallucinations, delusions, affective flattening, lack of motivation, social isolation and cognitive impairments. Features of the cognitive deficits observed in schizophrenic patients and in animal models of schizophrenia, such as attentional processes, cognition, working memory and behavioural flexibility, require the activation of the prelimbic cortex (PrL) and the ventral orbital cortex (VO), which are subregions of the prefrontal cortex. The thalamic reticular nucleus (Rt) also plays a key role in the regulation of these cortical functions by exerting an inhibitory control over the thalamo-cortical projections. The physiological role of a specific brain region could be associated with its gene expression profile. Moreover, the genes specifically expressed or enriched in one region could play a key role in the neuropathology of neuropsychiatric disorders. The aim of the project was two-fold: to identify genes enriched in three brain regions implicated in some aspects of cognition and neurobiological alteration observed in schizophrenia models (PrL, VO and Rt) compared to one control region (primary motor cortex. Ml); and to analyse the expression of selected enriched genes in a chronic PCP animal model of schizophrenia (Cochran et al. 2003). The expression profiling of the PrL, VO and Rt was performed using a combination of laser-assisted microdissection, linear amplification and GeneChip microarray analysis. The microdissections were performed on coronal sections dissected from 6 naive rat brains using the Leica AS LMD system. After extraction, the total RNA was linearly amplified, labelled and hybridised onto Affymetrix Rat Genome 230 2.0 arrays. The data were analysed according to the Rank Product method (Breitling et al, 2004). The regional overexpression of some selected genes was validated by in situ hybridisation (ISH) on six naive brains. The expression of these genes was then analysed by ISH after a chronic intermittent PCP treatment (2.58nig/kg) in six treated and six control rat brains. This study shows that the expression of many genes is differentially regulated in the selected areas. In the Rt, 429 transcripts were overexpressed compared to the cortical areas, Intra-cortical variations were also observed: 233 transcripts were overexpressed in the Prl compared to the other cortical areas, whereas 83 transcripts were enriched in the VO. The selectively expressed genes were involved in several biological mechanisms, such as neurotransmission, ion transport, neuronal development and synaptic plasticity. Ten genes were selected according to their levels of expression for the validation of both small and large differential expression by ISH. Expression analysis by ISH confirmed the overexpression in the PrL, VO and Rt predicted by the LMD-microarray for all genes tested. Wolframin, a gene which is likely to be involved in intracellular calcium trafficking that is the causative factor of Wolfram syndrome, showed significant decreased expression in the PrL in the PCP model of schizophrenia (p<0.05). Wolframin could be directly involved in the psychiatric conditions observed in the majority of Wolfram syndrome patients. Genome-wide expression analysis combined with laser-assisted microdissection showed that anatomical and functional differences between brain regions are associated with differential gene expression. Alterations of the region-specific expression could be crucial for psychiatric diseases; therefore this approach is exceptionally useful for the selection of candidate genes to study in models of psychiatric disorders such as schizophrenia. Using the LMD-microarray approach followed by ISH validation, this project defined the gene expression profiles of the Prl, VO and Rt in the rat brain and identified a novel gene with decreased expression in a chronic PCP model of schizophrenia.