Amphetamine-induced Conditioned Place Preference and Changes in mGlu1/5 Receptor Expression and Signaling in the Rat Medial Prefrontal Cortex

Abstract

The medial prefrontal cortex (mPFC) is implicated in the rewarding effect of psychostimulants, although molecular mechanisms underlying the rewarding properties of stimulants in this region are poorly understood. Group I metabotropic glutamate (mGlu) receptors (mGlu1/5 subtypes) are believed to be critical in this event. We thus in this study investigated changes in mGlu1/5 receptor expression and function in the rat mPFC in response to conditioned place preference (CPP) induced by amphetamine. Repeated amphetamine administration (2.5 mg/kg, once daily on alternate days for 10 days) induced reliable CPP. In the mPFC, surface expression of mGlu5 receptors was elevated in rats after amphetamine conditioning. mGlu5 receptors were also increased at synaptic and extrasynaptic sites in amphetamine-conditioned rats. Expression of mGlu1 receptors was stable in surface and synaptic compartments, while it was elevated in the extrasynaptic location. In mPFC neurons, the mGlu1/5 agonist-stimulated phosphoinositide signaling pathway was upregulated in its efficacy following amphetamine conditioning. The mGlu1/5 agonist-stimulated Src kinase phosphorylation was also augmented in rats treated with amphetamine. These results demonstrate the sensitivity of mPFC mGlu1/5 receptors to amphetamine-induced CPP. Amphetamine conditioning results in the upregulation of mGlu1/5 receptor expression at subcellular and/or subsynaptic levels and mGlu1/5-mediated postreceptor signaling in mPFC neurons.

Keywords: amphetamine; conditioned place preference; mGlu1; mGlu5; mPFC; metabotropic glutamate receptor.

Figure 1.. Repeated AMPH administration induces CPP in rats.

(A) Timeframe for an AMPH-induced CPP model. Following a 3-day pre-conditioning period (days 1–3), rats underwent a conditioning phase (days 4–13). Rats, after an AMPH injection (2.5 mg/kg, i.p.), were confined to one chamber. Same rats were treated with saline on alternate days and were confined to an opposite chamber. Three days (day 16) after the final conditioning injection, rats were tested for CPP and sacrificed for mPFC tissue collection. (B) Effects of AMPH on the time spent in the drug-paired chamber during pre- and post-conditioning tests. Note that AMPH significantly elevated the time spend in the paired chamber during a post-test (CPP test) relative to a pre-test. Data are presented as mean ± SEM (n = 9 in saline group, n = 11 in AMPH group). *P < 0.05 versus the pre-test.

Figure 2.. Effects of AMPH conditioning on surface and intracellular expression of mGlu1a/5 receptors in the rat mPFC.

(A and B) Effects of AMPH conditioning on surface and intracellular (Intra) expression of mGlu1a receptors and on the surface to intracellular (S:I) ratio of mGlu1a receptors. (C and D) Effects of AMPH conditioning on surface and intracellular expression of mGlu5 receptors and on the S:I ratio of mGlu5 receptors. Note that AMPH increased surface mGlu5 expression and the S:I ratio of mGlu5 receptors. Representative immunoblots (IB) are shown left to the quantified data (A and C). Rats were conditioned with AMPH (2.5 mg/kg, i.p.; once daily on alternate days for 10 days) or saline and were sacrificed 3 days after the last conditioning. Data are presented as means ± SEM (n = 9 in saline group, n = 11 in AMPH group). *P < 0.05 versus saline.

Figure 3.. Effects of AMPH conditioning on surface TfR and intracellular β-actin expression in the rat mPFC.

(A) Effects of AMPH conditioning on surface expression of TfRs. (B) Effects of AMPH conditioning on intracellular (Intra) expression of β-actin. Representative immunoblots (IB) are shown left to the quantified data. Rats were conditioned with AMPH (2.5 mg/kg, i.p.; once daily on alternate days for 10 days) or saline and were sacrificed 3 days after the last conditioning. Data are presented as means ± SEM (n = 9 in saline group, n = 11 in AMPH group).

Figure 4.. Effects of AMPH conditioning on synaptic and extrasynaptic expression of mGlu1a/5 receptors in the rat mPFC.

(A) Distribution of mGlu1a receptors in synaptic (Syn) and extrasynaptic (Extrasyn) compartments. (B) Distribution of mGlu5 receptors in synaptic and extrasynaptic compartments. (C and D) Effects of AMPH conditioning on synaptic versus extrasynaptic expression of mGlu1a (C) and mGlu5 (D) receptors. Note that AMPH increased extrasynaptic expression of mGlu1a receptors and synaptic and extrasynaptic expression of mGlu5 receptors. Representative immunoblots (IB) are shown left to (A and B) or above (C and D) the quantified data. Rats were conditioned with AMPH (2.5 mg/kg, i.p.; once daily on alternate days for 10 days) or saline and were sacrificed 3 days after the last conditioning. Data are presented as means ± SEM (n = 6 in saline group, n = 6 in AMPH group). *P < 0.05 versus saline.

Figure 5.. Effects of AMPH conditioning on DHPG-stimulated IP₃ production in the rat mPFC.

Note that the degree of the DHPG-induced IP₃ formation was augmented in AMPH-treated rats compared to saline-treated rats. Rats were conditioned with AMPH (2.5 mg/kg, i.p.; once daily on alternate days for 10 days) or saline and were sacrificed 3 days after the last conditioning for preparing mPFC slices. DHPG (50 μM) or vehicle was applied to slices for 20 s. Data are presented as means ± SEM (n = 9 in saline group, n =9 in AMPH group). *P < 0.05 versus vehicle.

Figure 6.. Effects of AMPH conditioning on DHPG-stimulated Src Y416 phosphorylation in the rat mPFC.

(A) Effects of DHPG on Src Y416 phosphorylation. B, Effects of AMPH conditioning on the DHPG-stimulated Y416 phosphorylation. Note that DHPG increased Y416 phosphorylation to a greater extent in AMPH- than saline-treated rats. Representative immunoblots (IB) are shown left to the quantified data. Rats were conditioned with AMPH (2.5 mg/kg, i.p.; once daily on alternate days for 10 days) or saline and were sacrificed 3 days after the last conditioning for preparing mPFC slices. DHPG at 100 (A) or 50 (B) μM was added to slices for 15 min prior to slice collections for Western blot. Data are presented as means ± SEM (n = 6 in saline group, n = 6 in AMPH group). *P < 0.05 versus vehicle.