Identification of genes regulated by chronic social stress in the rat dorsal raphe nucleus

Abstract

1. Changes in the serotonergic (5-HT) system are suspected to play a role in stress-induced neuropathologies and neurochemical measures indicate that serotonergic neurons in the dorsal raphe nucleus (DRN) are activated during stress. In the present study we analyzed gene expression in the DRN after chronic social stress using subtractive cDNA hybridization. 2. In the 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 hybridization was performed to clone sequences that are differentially expressed in the stressed animals. 3. From the cDNA libraries that were obtained, we selected the following genes for quantitative Real-time PCR: Two genes related to neurotransmission (synaptosomal associated protein 25 and synaptic vesicle glycoprotein 2b), a glial gene presumptively supporting neuroplasticity (N-myc downstream-regulated gene 2), and a gene possibly related to stress-induced regulation of transcription (CREB binding protein). These four genes were upregulated after the chronic social stress. Quantitative Western blotting revealed increased expression of synaptosomal associated protein 25 and synaptic vesicle glycoprotein 2b. 4. Genes directly related to 5-HT neurotransmission were not contained in the cDNA libraries and quantitative Real-time PCR for the serotonin transporter, tryptophan hydroxylase 2 and the 5-HT(1A) autoreceptor confirmed that these genes are not differentially expressed after 5-weeks of daily social stress. 5. These data show that 5 weeks of daily social defeat lead to significant changes in expression of genes related to neurotransmission and neuroplasticity in the DRN, whereas expression of genes directly related to 5-HT neurotransmission is apparently normal after this period of chronic stress.

Fig. 1.

Schematic drawing of the anatomical level at which the DRN was punched out. The caudal part of the brain was positioned on the specimen holder of a cryostat (at –18°C), and tissue was cut in caudal to rostral direction until −8.00 from bregma. DRN tissue was then punched out using a pre-cooled sharpened hypodermic needle (inner diameter 1.3 mm, −20°C) going 1 mm deep into the tissue (shaded areae). DRD, nucleus raphe dorsalis part dorsalis; DRV, nucleus raphe dorsalis part ventralis. DRVL, nucleus raphe dorsalis part ventrolateralis. Adapted from Paxinos and Watson, . Scale bar =1 mm.

Fig. 2.

Effect of chronic social stress on expression of genes in the DRN: Synaptosomal associated protein 25 kD (SNAP-25), synaptic vesicle glycoprotein 2b (SV2b), CREB binding protein (CBP) and N-myc down-stream regulated gene 2 (NDRG2). Data were generated with Real-time PCR. Transcript expression is presented as percentage of mRNA for the housekeeping gene GAPDH. Significant differences between stressed and control animals are indicated by asterisks (n=8/group; two-tailed unpaired Student's t-test; ^* p<0.05;^** p<0.01). SV2b showed a trend towards increased expression in stressed animals (p=0.051). Data are expressed as mean±SEM.

Fig. 3.

Expression of serotonin related genes, serotonin transporter (SERT), 5-HT_1A autoreceptor (5-HT_1A) and tryptophan hydroxylase 2 (TPH2) after chronic social stress. Data were generated by Real-time PCR. Transcript expression is presented as percentage of mRNA for the housekeeping gene GAPDH (n=8/group). No significant differences were found. Data are expressed as mean±SEM.

Fig. 4.

Western blot analysis revealed that chronic social stress (5 weeks) increases expression of (A) synaptosomal associated protein 25 (SNAP-25) and (B) synaptic vesicle glycoprotein 2b (SV2b). Equal amount of total proteins from pooled DRN of controls (lane 1) and stressed animals (lane 2) were loaded. An anti-ß-actin antibody was used for control (upper panel in A & B). For each protein, the relative optical density (OD) of the bands was determined and presented as percentage of the respective band for the control animals (n=8/group). Results were obtained from 3 independent experiments for each protein and presented as mean±SEM. Two-tailed unpaired Student's t-test was performed; ^* p<0.05. The difference in the SNAP-25 protein expression did not reach statistical significance (p=0.065).