Although EAAT may not be involved in the

Although EAAT3 may not be involved in the induction of morphine-induced CPP, our results suggest that other EAATs may play a role in the behavior because riluzole, an EAAT activator (Frizzo et al., 2004, Fumagalli et al., 2008), abolished morphine-induced CPP. Consistent with this suggestion, a few studies have shown that morphine reduces EAAT1 and EAAT2 expression in the central nervous system (Ozawa et al., 2001, Mao et al., 2002). Our results showed that EAAT3 expression in the plasma membrane was increased in the mPFC, nucleus accumbens and VTA but not in the hippocampus at 24h after the last dose of morphine. This result suggests region-specific regulation of EAAT3 by morphine. However, future studies are needed to identify the mechanisms for this regulation. Nevertheless, mPFC, nucleus accumbens and VTA are important TNF-alpha, recombinant murine protein regions for drug addiction (Shah and Treit, 2004, Jasinska et al., 2014, Rosen et al., 2015, Sadeghzadeh et al., 2016); whereas the hippocampus is a critical brain region for learning and memory. These results may not mean that learning and memory are not involved in morphine addiction but suggest that EAAT3 among many factors in the hippocampus may not be important for morphine addiction. VTA and its projections to the nucleus accumbens have been shown to be the primary neural structures for the acute rewarding behaviors of opioids (Wise, 1989, Rosen et al., 2015). PFC is implicated in opioid-related learning and memory and receives inputs from VTA (Daglish et al., 2001, Rosen et al., 2015). Dopamine and glutamate neurotransmission in these brain regions plays an important role in these learning and memory functions (D'Souza, 2015, Rosen et al., 2015). Our finding that EAAT3 may participate in delaying extinction of morphine-induced CPP suggests that glutamate neurotransmission in the related structures contributes to the extinction of morphine addiction. Our findings may have significant implications. There is no effective intervention for morphine addiction. Our study suggests that riluzole may have a great therapeutic potential for preventing morphine addiction. This finding may be easy for clinical translation because riluzole has already been used in clinic for neurodegenerative diseases (Cheah et al., 2010). Our data also suggest that EAAT3 may regulate the extinction of morphine-induced CPP. This finding is in agreement with our previous results that EAAT3 may regulate learning and memory (Lee et al., 2012, Cao et al., 2014, Wang et al., 2014) and add additional evidence for its role in learning and memory. In addition, our results suggest that EAAT3 can be a target for developing interventions to speed up the recovery from morphine addiction. Our study has limitations. We used riluzole to determine the possible role of EAATs in morphine-induced CPP. However, riluzole is a general EAAT activator. Thus, it is not clear which types of EAATs may play a role in the morphine-induced CPP. In addition, riluzole has other effects, such as inhibition of glutamate release (Wang et al., 2004). These effects may be relevant to the effects because glutamate receptor plasticity has been shown to play a role in morphine sensitization (Xia et al., 2011). It is not clear whether these effects may contribute to its inhibition of morphine-induced CPP. Nevertheless, our major goal of this study was to determine the role of EAAT3 in the morphine-induced CPP. Currently, specific inhibitor or activator of EAAT3 has not been identified yet. It is not possible to perform a study using a specific EAAT3 inhibitor or activator to support our findings from EAAT3 knockout mice.
Statement of interest
Author contributions
Acknowledgments This study was supported by grants (R01 GM065211 and R01 GM098308 to Z Zuo) from the National Institutes of Health, Bethesda, Maryland, by a grant from the International Anesthesia Research Society (2007 Frontiers in Anesthesia Research Award to Z Zuo), Cleveland, Ohio, by a Grant-in-Aid from the American Heart Association Mid-Atlantic Affiliate (10GRNT3900019 to Z. Zuo), Baltimore, Maryland, and the Robert M. Epstein Professorship Endowment, University of Virginia.