Evidence for the involvement of ERbeta and RGS9-2 in 17-beta estradiol enhancement of amphetamine-induced place preference behavior

Abstract

Estrogen enhances dopamine-mediated behaviors, which make women and female rats more sensitive to the effects of the psychostimulant drugs, cocaine and amphetamine. How cocaine and amphetamine elicit more robust behavioral responses in females remains unclear, but studies have shown that the Regulator of G-protein Signaling 9-2 (RGS9-2) protein is an important modulator of the behavioral responses to these drugs. Previously, we reported that 17-beta estradiol reduced RGS9-2 mRNA expression in the shell of the nucleus accumbens, but not the core. The present studies were designed to further evaluate the involvement of RGS9-2 in estradiol enhancement of amphetamine-induced place preference behavior and to examine which estrogen receptor subtype mediates the effect of estradiol. Female Sprague-Dawley rats were ovariectomized and treated for 14 days with an inert vehicle or 17-beta estradiol (by Silastic implant or injection [80 microg/kg]). 17-beta-Estradiol-treated female rats had enhanced amphetamine-induced conditioned place preference behavior compared to vehicle-treated, ovariectomized female rats. In situ hybridization histochemistry and Western blotting identified an inverse relationship between RGS9-2 protein expression in the nucleus accumbens shell and the hormonal enhancement of amphetamine-induced place preference behavior. A similar relationship was not found between place preference behavior and RGS9-2 expression in the accumbens core. Moreover, treatment of ovariectomized female rats with the selective estrogen receptor-beta agonist, diarylpropionitrile (1 mg/kg), for 2 weeks also facilitated amphetamine-induced place preference behavior and selectively reduced nucleus accumbens shell RGS9-2 protein expression. These data provide insight into a potential mechanism by which estrogen and/or sex modulate mesoaccumbal dopamine receptor signaling and possibly, addictive behaviors.

Figure 1. Histological Example of Nucleus Accumbens

A representative coronal section depicting the NAc shell and core. The scale bar is 1-mm. The 1-mm micropunches extracted from the slice are represented by a filled circle (shell) and an open circle (core).

Figure 2

Figure 2A. Place Preference to AMPH in OVX rats with and without Gonadal Hormone Replacement The effects of OVX, OVX+E2 and OVX+E2+P replacement on AMPH-induced CPP in female rats are shown. Data are represented as the mean (±S.E.M.) time spent in the drug paired chamber minus the time spent in the saline paired chamber (CPP scores). * indicates a statistically significant difference from OVX controls (p<0.05 by one-way ANOVA and Tukey’s post-hoc comparison). There were 5 female rats in the OVX, OVX+E2 and OVX+E2+P groups. Figure 2B. Effects of Gonadal Hormones on RGS9-2 Following AMPH-induced CPP in the NAc Shell Data are presented as the mean (±S.E.M.) integrated density of RGS9-2 protein expression in the NAc shell of OVX, OVX+E2 and OVX+E2+P treated females sacrificed after CPP testing. There were 5 rats in each treatment group. Significant reductions in RGS9 expression in the NAc shell are indicated by * (p<0.05) or **(p<0.01). Inset of figure shows a representative Western blot of RGS9-2 expression in the NAc Shell using 15 micrograms of tissue extracted utilizing micropunches. RGS9-2 is a 78 kDa protein shown in the upper image. The lower image shows beta-actin expression (43 kDa) expression. Figure 2C. Effects of Gonadal Hormones on RGS9-2 Following AMPH-induced CPP in the NAc Core Quantification of the RGS9-2 expression in of the NAc core tissue. Data are presented as the mean (±S.E.M.) of integrated density. The inset of the figure displays a representative immunoblot for Experiment 1 on NAc core samples. There were 5 rats in each treatment group.

Figure 3

Figure 3A. The Effects of OVX and Selective Agonists on AMPH-induced CPP in Female Rats Place preference is expressed by the mean (±S.E.M.) time spent in the drug paired chamber minus the time spent in the saline paired chamber (CPP scores). * indicates a statistically significant difference compared to OVX control rats (p<0.05) by one-way ANOVA and Tukey’s post-hoc comparison. There were 9 animals in the OVX, OVX+E2, OVX+PPT and OVX+DPN groups. Figure 3B. Effects of Selective Agonists on RGS9-2 expression in the NAc Shell Data are presented as the mean (±S.E.M.) integrated density of RGS9-2 protein expression in the NAc shell of the four groups OVX, OVX+E2, OVX+PPT and OVX+DPN (n=4/group). Significantly reductions in RGS9-2 expression in the NAc shell are indicated by * (p<0.05). Figure 3C. Representative Immunoblots from Experiment 2 in the NAc Shell Expression of RGS9-2 in the NAc shell using 15 micrograms of tissue extracted utilizing micropunches. The upper image shows expression of the 78 kDa protein, RGS9-2. The lower image shows beta-actin expression (43 kDa) expression. Lane 1 depicts RGS9-2 protein expression in the NAc shell of OVX female rat. Lanes 2 shows expression in an OVX+E2 female rat. Lane 3 depicts expression in an OVX+PPT treated female rat and lane 4 depicts expression in a DPN-treated OVX female rat.