Several studies have shown that

Several studies have shown that regulation of glutamate homeostasis is critical in relapse to many drugs of abuse [84,85]. Glutamate is transported by GLT-1, which accounted for about 90% of glutamate clearance from the synaptic cleft [14,15]. CEF is known to produce its effects through GLT-1 upregulation. However, in this study, we found that CEF treatment was not associated with changes in GLT-1 expression in all tested Cilnidipine australia areas. While no change in GLT-1 expression has been shown, there is a great possibility that CEF could improve the function of GLT-1 without changing its expression. Further studies are warranted to determine the activity of GLT-1 after CEF treatment in HYD-seeking behavior, for example, measuring extracellular glutamate concentrations using microdialysis technique. Another possibility is that the expression of GLT-1 might be different between sub-regions, where the effect in one area could be masked by the other. Future studies are still needed to determine sub-region differential effects in GLT-1 expression. Studies have shown that chronic exposure to morphine reduced the expression of GLT-1 mRNA in the NAc, striatum and thalamus [7]. In addition, most of morphine studies, with regard to the glutamatergic system, were investigated in the cerebellum and spinal cord, which are more related to pain management, but not to relapse [[86], [87], [88]]. Thus, studies are required to investigate the effects of HYD with different doses and strains as well to examine the relationship between GLT-1 expression and other glutamate transporters in HYD exposure and reinstatement. Although no changes were observed in GLT-1 expression with HYD reinstatement, we found that the xCT expression was downregulated in the NAc and HIP. Cellular mechanistic events involve xCT, GLT-1 and other glutamatergic receptors in several brain regions (e.g. NAc and HIP) to modulate HYD-seeking behavior are demonstrated in (Fig. 8). To the best of our knowledge, little is known about the function of xCT in opioid dependence and relapse. However, it was reported that N-acetylcysteine can restore the function of xCT system and attenuate drug-seeking behavior in animals [89]. Dysfunction of xCT can occur after cocaine self-administration, which was associated with a reduction in basal glutamate concentration in the NAc [90,91]. Similarly, repeated morphine exposure was associated with low basal glutamate concentration in the HIP in mice [92]. It is important to note that restoring the xCT function can attenuate cocaine [90,91] and heroin seeking behavior [25]. Moreover, it has been shown that repeated administration of morphine can lead to behavioral sensitization in animals. Thus, morphine-sensitized rats have been shown to have high level of extracellular glutamate in the HIP, when they were challenged with morphine after prolonged abstinence [93]. Therefore, restoring xCT function was assumed to improve the glutamatergic tone on mGluR2/3 and attenuate the seeking behavior as previously reported in cocaine [26]. Here, we assumed that the downregulation of xCT expression in the NAc and HIP might be associated with reinstatement to HYD in P rats. Therefore, restoring xCT expression in these brain regions with CEF could, in part, attenuate HYD reinstatement (Fig. 8). Furthermore, our study investigated another glial glutamate transporter called GLAST. GLAST expression was not changed in HYD reinstatement in all tested groups. This is in accordance with a previous study, which demonstrated that GLAST mRNA expression was not altered after morphine administration in the thalamus, hypothalamus, cerebral cortex, HIP, striatum, midbrain, cerebellum, and pons-medulla [7]. Although other studies have shown that morphine administration is associated with reductions in GLAST and EAAT3 expression in the spinal cord [18,19]. In fact, GLAST is highly expressed in the cerebellum and spinal cord and less expressed in other brain regions [17]. Together, these data suggest that GLAST is less involved in HYD reinstatement.