Identification of Genes in the Dorsal Raphe Nucleus Regulated by Chronic Stress and Citalopram

It is known that stress, especially when it is chronic, can lead to mood disorders and changes in the serotonergic (5-HT) system, which most probably play a role in stress-induced neuropathologies. Neurochemical measures indicated that serotonergic neurons in the dorsal raphe nucleus (DRN) are activated by stress. Clinical studies have shown that the therapeutic effect of antidepressants, including specific serotonin re-uptake inhibitors (SSRIs), occurs only after 2-3 weeks of chronic treatment. This lag phase may indicate that antidepressants change central nervous gene expression in a time dependent manner, however, little is known about such processes in stressed subjects. The aim of the present thesis was to identify genes in the rat DRN that are regulated by chronic social stress and to investigate whether these genes are regulated by the selective SSRI citalopram.The first part of the thesis describes the identification of genes that are differentially expressed by chronic social stress in the DRN. Using a resident intruder paradigm, male Wistar rats were chronically stressed by daily social defeat during 5 weeks. RNA was isolated from their DRN, cDNA was generated, and subtractive cDNA hybridization was performed to clone sequences that are differentially expressed in the stressed animals. From the cDNA libraries that were obtained, the following genes were selected to quantify mRNA expression using quantitative real-time PCR: Two genes related to neurotransmission, synaptosomal associated protein-25 (SNAP-25) and synaptic vesicle glycoprotein 2b (SV2b); a glial gene presumptively supporting neuroplasticity (N-myc downstream-regulated gene 2, NDRG2); neuron specific enolase (NSE) that is known to promote neuronal growth and survival; and a gene possibly related to stress-induced regulation of transcription, CREB binding protein (CBP). Expression of mRNA for these genes was significantly upregulated in the DRN of chronically stressed rats. Genes directly related to 5-HT neurotransmission could not be identified by subtractive cDNA hybridization which may suggest that their expression is not regulated by stress. However, quantitative real-time PCR was nevertheless performed for the serotonin transporter (SERT), the 5-HT1A autoreceptor and the tryptophan hydroxylase (TPH) genes 1 and 2. It was found that only TPH1 mRNA was upregulated by chronic stress. These data reveal that 5 weeks of daily social defeat leads to significant changes in the expression of genes related to neurotransmission, neuroplasticity and 5-HT synthesis in the DRN, whereas expression of genes directly related to 5-HT release is apparently normal after this period of chronic stress.The second part of the thesis was designed to investigate the impact of a chronic citalopram treatment (30mg/kg/day, for 4 weeks administered via drinking water) on the mRNA expression of the above described genes in the DRN. The appropriate dose of citalopram was determined in a pilot study. Real-time PCR showed that citalopram normalized the stress-induced upregulation of mRNA for three genes: SV2b, CBP and NDRG2. The SSRI had no significant effect on SNAP-25 mRNA, but upregulated the expression of NSE mRNA in both stressed and unstressed animals. The potential impact of citalopram on the genes directly related to serotonin transmission was also investigated. It was found that citalopram reduced 5-HT1A autoreceptor mRNA expression only in stressed animals. TPH 1 and 2 genes respond differentially to citalopram. The expression of TPH1 mRNA was normalized only in the stressed animals, whereas TPH2 mRNA was reduced in all treated subjects. These findings demonstrate that in the DRN of chronically stressed rats, citalopram restores mRNA expression of distinct genes involved in neurotransmitter release/ neuroplasticity and 5-HT biosynthesis.To analyze whether stress and citalopram also change protein expression in the DRN, Western blot experiments were performed. These experiments revealed that chronic stress increased the expression of SV2b, SNAP-25, NSE, and TPH protein. Citalopram reversed the stress-induced upregulation of SV2b but had no significant effect on the amount of SNAP-25 protein. Furthermore, expression of syntaxin 1A and synaptophysin protein was not affected by either stress or citalopram. This indicates that stress and citalopram have no global effect on all synaptic/synaptic vesicle proteins in the DRN but that only distinct genes are affected. Stress-induced upregulation of NSE protein was not reversed by the SSRI but instead, citalopram enhanced NSE expression in control animals. The stress-induced upregulation of TPH protein correlated with enhanced expression of TPH1 mRNA, whereas antidepressant-induced normalization of TPH protein expression appears to be due to the reduction in TPH 1 and 2 mRNA.To investigate whether the effects of stress and citalopram on gene expression are confined to the DRN, protein expression was also determined in the hippocampal formation. Data indicated that also in this brain region NSE expression is involved in stress-induced processes, however, citalopram had no effect on hippocampal NSE expression. In contrast to the DRN, hippocampal syntaxin 1A protein was upregulated by stress and normalized by citalopram. In conclusion, the present data demonstrate that chronic stress upregulates the expression of distinct genes involved in neurotransmitter release/neuroplasticity which possibly reflect enhanced neuronal activity. The SSRI citalopram normalizes expression of some of these genes. Furthermore, the changes in gene expression are specific for the DRN indicating a regional effect of stress and citalopram, respectively. The stress-induced upregulation of TPH may reflect enhanced activity of 5-HT neurons, while the SSRI normalized TPH expression. One may speculate that similar mechanisms may contribute to the therapeutic actions of citalopram in patients.