Glutamate transporter type 3 participates in maintaining morphine-induced conditioned place preference

Abstract

Glutamate transporters (EAAT) have been implicated in the drug addiction behavior. We determined whether EAAT type 3 (EAAT3) played a role in morphine addiction. Six- to eight-week-old EAAT3 knockout (EAAT3^-/-) mice and their wild-type littermates received 3 intraperitoneal injections of 10mg/kg morphine, each on an alternative day, to induce conditioned place preference (CPP). Two days after the place preference returned to baseline, mice received 2.5mg/kg morphine to induce reinstatement. Some mice received intraperitoneal injection of 4mg/kg riluzole, an EAAT activator, 30min before morphine or saline injection. Hippocampus, medial prefrontal cortex, nucleus accumbens and ventral tegmental area were harvested for Western analysis 24h after the last dose of morphine was injected. Morphine induced CPP in wild-type and EAAT3^-/- mice. Gender is not a statistically significant factor to influence this behavior. This conditioned behavior extinguished after morphine administration was stopped for 8-9days in wild-type mice, while this extinction occurred 6days after discontinuation of morphine injection in EAAT3^-/- mice. A small dose of morphine similarly reinstated the conditioned behavior in the wild-type and EAAT3^-/- mice. Riluzole abolished morphine-induced CPP during the initial place preference. Morphine increased EAAT3 expression in the plasma membrane of medial prefrontal cortex, nucleus accumbens and ventral tegmental area but did not affect EAAT3 expression in the hippocampus. These results suggest that EAAT3 delays the extinction of morphine-induced CPP. EAAT activation may prevent the formation of morphine-induced CPP.

Keywords: conditioned place preference; glutamate transporter; mice; morphine.

Fig. 1

Diagram of experiments.

Fig. 2

Morphine-induced CPP. Baseline place preference was tested on day −3 to −1. Mice received 10 mg/kg morphine or saline on day 0, 2 and 4. All mice received saline on day 1, 3 and 5 and did not receive any injections on the following days when they were tested for place preference extinction. A: wild-type mice. B: EAAT3^−/− mice. Results are mean ± S.E.M. (n = 8 − 13). * P < 0.05 compared with the corresponding data on day −1. ^ P < 0.05 compared with corresponding data of male mice receiving saline. # P < 0.05 compared with corresponding data of female mice receiving saline.

Fig. 3

Morphine-induced increase of EAAT3 expression. A: medial prefrontal cortex was collected from wild-type (WT) and EAAT3^−/− mice for Western analysis. B to E: Animals received 3 doses of 10 mg/kg morphine, each on alternative days. Brain tissues were harvested at 24 h after the last dose of morphine. Representative images of Western blots are presented in panel B and D. Quantitative results are in panel C and E (mean ± S.E.M., n = 8). * P < 0.05 compared with mice receiving saline.

Fig. 4

Reinstatement of morphine-induced CPP. Two days after the extinction of morphine-induced CPP, mice whose CPP data are shown in figure 2 received 2.5 mg/kg morphine or saline. Results are mean ± S.E.M. (n = 8 − 13). * P < 0.05 compared with mice receiving saline.

Fig. 5

Riluzole abolished morphine-induced CPP. Baseline place preference was tested on day −3 to −1. Mice received 10 mg/kg morphine or saline on day 0, 2 and 4. All mice received saline on day 1, 3 and 5. Some mice received riluzole 30 min before each injection of morphine or saline. Results are mean ± S.E.M. (n = 7 − 8). * P < 0.05 compared with the corresponding data on day −1.