Heightened transcription for enzymes involved in norepinephrine biosynthesis in the rat locus coeruleus by immobilization stress

Background: The locus coeruleus (LC), a target for CRH neurons, is critically involved in responses to stress. Various physiological stresses increase norepinephrine turnover, tyrosine hydroxylase (TH) enzymatic activity, protein and mRNA levels in LC cell bodies and terminals; however, the effect of stress on other enzymes involved in norepinephrine biosynthesis in the LC is unknown. Methods: Rats were exposed to single (2 hour) or repeated (2 hour daily) immobilization stress (IMO). Recombinant rat dopamine b–hydroxylase (DBH) cDNA was expressed in E. coli and used to generate antisera for immunohistochemistry and immunoblots in LC. Northern blots were used to assess changes in mRNA levels for TH, DBH, and GTP cyclohydrolase I (GTPCH) in the LC in response to the stress. Conditions were found to isolate nuclei from LC and to use them for run–on assays of transcription. Results: Repeated stress elevated the DBH immunoreactive protein levels in LC. Parallel increases in TH, DBH and GTPCH mRNA levels of about 300% to 400% over control levels were observed with single IMO, and remained at similar levels after repeated IMO. This effect was transcriptionally mediated, and even 30 min of a single IMO significantly increased the relative rate of transcription. Conclusions: This study is the first to reveal transcriptional activation of the genes encoding catecholamine biosynthetic enzymes in the LC by stress. In addition to TH, changes in DBH and GTPCH gene expression may also contribute to the development of stress–triggered affective disorders.