Regulation of tyrosine hydroxylase promoter activity by chronic morphine in TH9.0-LacZ transgenic mice

Abstract

Levels of tyrosine hydroxylase (TH), the rate-limiting enzyme in catecholamine biosynthesis, are known to be upregulated in specific brain regions by chronic administration of drugs of abuse. Chronic morphine administration increases TH levels in the locus coeruleus and ventral tegmental area, whereas chronic cocaine administration increases TH levels in the ventral tegmental area only. While such upregulation of TH has been related to behavioral effects of the drugs, the mechanism underlying these adaptations has remained controversial. To study the possibility that upregulation of TH occurs at the transcriptional level, we investigated the effect of chronic morphine or cocaine treatment on the activity of the TH gene promoter (9.0 kb), coupled to the LacZ reporter gene, in transgenic mice. These TH9.0-LacZ mice have been shown to exhibit correct tissue-specific expression and regulation of the reporter gene. We show here that chronic (but not acute) exposure of the TH9.0-LacZ mice to morphine increases the expression of beta-galactosidase (which is encoded by the LacZ gene) in the locus coeruleus by twofold compared with sham-treated mice. In contrast, beta-galactosidase expression in the ventral tegmental area was decreased 20-25% by chronic morphine and unaffected by chronic cocaine administration. Similar results were obtained after analysis of TH mRNA levels in these brain regions by in situ hybridization. These results suggest that chronic morphine upregulates TH expression via transcriptional mechanisms in the locus coeruleus but by post-transcriptional mechanisms in the ventral tegmental area.

Fig. 1.

Expression of β-galactosidase in the LC of TH9.0-LacZ mice after chronic treatment with morphine. Low-power photomicrographs of representative brain slices through the LC of sham-treated mice (A, C,E, G) or chronic morphine-treated mice (B, D, F,H) are shown. Expression of β-galactosidase was visualized with X-gal histochemistry, performed as described in Materials and Methods, with reaction times of 15 min (A,B), 1 hr (C, D), 4 hr (E, F), or 16 hr (G, H).

Fig. 2.

Quantitative analysis of β-galactosidase expression in the LC of TH9.0-LacZ mice after chronic treatment with morphine. Mice received sham treatment (n = 4) or chronic morphine treatment (n = 4), and β-galactosidase expression was visualized, as described in Materials and Methods. Digital analysis and densitometry were performed on the LC of slide-mounted brain sections (see Materials and Methods). Data shown are the means ± SD. **p = 0.01; ***p = 0.001, compared with sham-treated mice byt test.

Fig. 3.

Expression of β-galactosidase in the VTA of TH9.0-LacZ mice after chronic treatment with morphine. Mice received sham treatment (n = 4) or chronic morphine treatment (n = 4), and β-galactosidase expression was visualized with X-gal histochemistry, performed as described in Materials and Methods. Low-power photomicrographs of representative brain slices through the VTA of sham-treated mice (A) or morphine-treated mice (B) are shown (for 16 hr X-gal reaction time).C, Quantitative analysis of β-galactosidase expression was performed on the VTA region of slide-mounted brain sections for 15 min, 1 hr, 4 hr, or 16 hr X-gal reaction times (see Materials and Methods). Data shown are the means ± SD. *p = 0.05, compared with sham mice by t test.

Fig. 4.

Regulation of TH immunoreactivity in the mouse VTA after chronic treatment with morphine. TH9.0-LacZ mice received chronic sham (S) or morphine (M) treatment, and levels of TH immunoreactivity were determined by Western blotting, as described in Materials and Methods. Representative autoradiograms are shown. Data are expressed as mean percent of sham ± SEM (n = 6); *p < 0.001 byt test.

Fig. 5.

Expression of TH mRNA in the rat VTA after chronic treatment with morphine. Rats received chronic sham (S) or morphine (M) treatment, and levels of TH mRNA were determined by in situ hybridization, as described in Materials and Methods. Low-power autoradiographs of representative brain slices through the VTA (left) and LC (right) are shown. The figure is representative of results obtained from six to nine animals in each treatment group.