Stress upregulates TPH1 but not TPH2 mRNA in the rat dorsal raphe nucleus: identification of two TPH2 mRNA splice variants

Abstract

Serotonin is implicated in stress-related psychopathologies. Two isoforms of the rate-limiting enzyme of serotonin biosynthesis, tryptophan hydroxylase, TPH1 and TPH2, are known. We show here that in the rat dorsal raphe nucleus (DRN), the nucleus that contains the highest number of 5-HT neurons in the brain, TPH1 mRNA reveals a low level of expression but is detectable both by quantitative real-time PCR and in situ hybridization whereas in the pineal gland (PiG), TPH1 mRNA is strongly expressed. To examine effects of stress on TPH expression we exposed male Wistar rats to daily restraint stress for 1 week. As shown by quantitative real-time PCR, TPH1 mRNA is 2.5-fold upregulated by the stress in DRN but not in PiG. Using 3'-RACE, we identified two TPH2 mRNA splice variants in the rat DRN which differ in the length of their 3'-untranslated regions (UTRs). TPH2b (with a short 3'-UTR) is the predominant variant in the DRN, whereas TPH2a (with a longer 3'-UTR) shows a low abundance in this nucleus. In the PiG, only TPH2b is detectable revealing a low level of expression. Expression of both TPH2 splice variants is not affected by stress, neither in DRN nor in the PiG. These data indicate that TPH1 in the serotonergic neurons of the DRN might be relevant for stress-induced psychopathologies.

Fig. 1

3′-RACE analysis of TPH2 transcripts in the dorsal raphe nucleus. (a) 3′-RACE resulted in two bands (about 1,320 and 1,050 bp; lane 1). Negative controls for the 3′-RACE PCR included: control with oligo dT primer only (lane 2), control with gene-specific primer only (lane 3) or both primers were added while the RACE-ready cDNA omitted (lane 4). (b) A schematic map of TPH2a and TPH2b splice variants showing the length of the 3′-UTRs and the polyadenylation sites

Fig. 2

Expression of TPH mRNA in the dorsal raphe nucleus and the pineal gland. Data were generated by quantitative Real-time PCR with transcript expression being normalized to the GAPDH housekeeping gene. Repeated stress increases TPH1, but not TPH2a and TPH2b mRNA in the DRN (left). No effect of stress on either TPH mRNA was found in the PiG (right). Significant difference between stress and control is indicated by asterisks (Student’s t-test, **, P < 0.01). Data are expressed as mean ± SEM

Fig. 3

In situ hybridization for TPH1 with brain stem sections from the level of dorsal raphe nucleus (DRN). Autoradiograms showing in situ hybridization with the ³³P-UTP labeled TPH1 riboprobe on brain stem sections of a control (a) and a stressed rat (b). Hybridization with the sense probe is shown (c). A Nissl stained section from the same neuroanatomical level is also presented (d). Note the hybridization signal in the DRN (arrow) of stressed rats (in b). Scale bar: 1 mm. PiG, pineal gland