Gene expression profiling following short-term and long-term morphine exposure in mice uncovers genes involved in food intake

Abstract

Addictive drugs including opioids activate signal transduction pathways that regulate gene expression in the brain. However, changes in CNS gene expression following morphine exposure are poorly understood. We determined changes in gene expression following short- and long-term morphine treatment in the hypothalamus and pituitary using genome-wide DNA microarray analysis and confirmed those alterations in gene expression by real-time reverse transcriptase polymerase chain reaction (RT-PCR) analysis. In the hypothalamus, short-term morphine administration up-regulated (at least twofold) 39 genes and down-regulated six genes. Long-term morphine treatment up-regulated 35 genes and down-regulated 51 genes. In the pituitary, short-term morphine administration up-regulated 110 genes and down-regulated 29 genes. Long-term morphine treatment up-regulated 85 genes and down-regulated 37 pituitary genes. Microarray analysis uncovered several genes involved in food intake (neuropeptide Y, agouti-related protein, and cocaine and amphetamine-regulated transcript) whose expression was strongly altered by morphine exposure in either the hypothalamus or pituitary. Subsequent RT-PCR analysis confirmed similar regulation in expression of these genes in the hypothalamus and pituitary. Finally, we found functional correlation between morphine-induced alterations in food intake and regulation of genes involved in this process. Changes in genes related to food intake may uncover new pathways related to some of the physiological effects of opioids.

Fig. 1

Four day, but not six h morphine pellet administration leads to opioid dependence as determined by abstinence withdrawal. Mice were implanted with a placebo [6 h (n=3) or 4 days (n=4)] or morphine [6 h (n=6) or 4 days (n=5)] pellet. The pellets were removed after 6 h or 4 days and mice were observed for signs of opiate withdrawal that was determined by counting the number (± SEM) of jumps (A), rearing (B) and forepaw tremors (C) over the next two h following pellet removal. The 6 h and 4 day placebo groups were pooled. *** P<0.001 vs placebo pellet, ### P<0.001 vs 6 h morphine pellet.

Fig. 2

Food intake (A), average weight change from baseline (B), NPY expression (C) and AgRP (D) expression in mice treated for four days with morphine or placebo pellets or placebo pellets pair-fed to match food intake on the prior day of morphine pellet-mice. Pair-feeding occurred on days 1–4 and morphine mice are shifted by one day so that their food intake is aligned with that of pair-fed mice. Animals were weighed daily and weight change from baseline was calculated. The amount of food consumed per mouse was determined daily. Baseline food intake and weight were measured for two days prior to pellet implantation in placebo- and pair-fed animals and for one day in morphine-treated animals. Hypothalamic NPY and AgRP expression was determined by real-time RT-PCR and corrected for expression of 18S RNA. Food intake, N=8–11 animals, body weight, N=10–12 animals, Gene expression, N=7–14 animals. *, P< 0.05, **, P< 0.01, ***, P< 0.001, ****, P< 0.0001.

Fig. 2

Food intake (A), average weight change from baseline (B), NPY expression (C) and AgRP (D) expression in mice treated for four days with morphine or placebo pellets or placebo pellets pair-fed to match food intake on the prior day of morphine pellet-mice. Pair-feeding occurred on days 1–4 and morphine mice are shifted by one day so that their food intake is aligned with that of pair-fed mice. Animals were weighed daily and weight change from baseline was calculated. The amount of food consumed per mouse was determined daily. Baseline food intake and weight were measured for two days prior to pellet implantation in placebo- and pair-fed animals and for one day in morphine-treated animals. Hypothalamic NPY and AgRP expression was determined by real-time RT-PCR and corrected for expression of 18S RNA. Food intake, N=8–11 animals, body weight, N=10–12 animals, Gene expression, N=7–14 animals. *, P< 0.05, **, P< 0.01, ***, P< 0.001, ****, P< 0.0001.

Fig. 2

Food intake (A), average weight change from baseline (B), NPY expression (C) and AgRP (D) expression in mice treated for four days with morphine or placebo pellets or placebo pellets pair-fed to match food intake on the prior day of morphine pellet-mice. Pair-feeding occurred on days 1–4 and morphine mice are shifted by one day so that their food intake is aligned with that of pair-fed mice. Animals were weighed daily and weight change from baseline was calculated. The amount of food consumed per mouse was determined daily. Baseline food intake and weight were measured for two days prior to pellet implantation in placebo- and pair-fed animals and for one day in morphine-treated animals. Hypothalamic NPY and AgRP expression was determined by real-time RT-PCR and corrected for expression of 18S RNA. Food intake, N=8–11 animals, body weight, N=10–12 animals, Gene expression, N=7–14 animals. *, P< 0.05, **, P< 0.01, ***, P< 0.001, ****, P< 0.0001.

Fig. 2

Food intake (A), average weight change from baseline (B), NPY expression (C) and AgRP (D) expression in mice treated for four days with morphine or placebo pellets or placebo pellets pair-fed to match food intake on the prior day of morphine pellet-mice. Pair-feeding occurred on days 1–4 and morphine mice are shifted by one day so that their food intake is aligned with that of pair-fed mice. Animals were weighed daily and weight change from baseline was calculated. The amount of food consumed per mouse was determined daily. Baseline food intake and weight were measured for two days prior to pellet implantation in placebo- and pair-fed animals and for one day in morphine-treated animals. Hypothalamic NPY and AgRP expression was determined by real-time RT-PCR and corrected for expression of 18S RNA. Food intake, N=8–11 animals, body weight, N=10–12 animals, Gene expression, N=7–14 animals. *, P< 0.05, **, P< 0.01, ***, P< 0.001, ****, P< 0.0001.