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, 39 (8), 1833-42

Amphetamine Self-Administration Attenuates Dopamine D2 Autoreceptor Function

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Amphetamine Self-Administration Attenuates Dopamine D2 Autoreceptor Function

Erin S Calipari et al. Neuropsychopharmacology.

Abstract

Dopamine D2 autoreceptors located on the midbrain dopaminergic neurons modulate dopamine (DA) neuron firing, DA release, and DA synthesis through a negative-feedback mechanism. Dysfunctional D2 autoreceptors following repeated drug exposure could lead to aberrant DA activity in the ventral tegmental area (VTA) and projection areas such as nucleus accumbens (NAcc), promoting drug-seeking and -taking behavior. Therefore, it is important to understand molecular mechanisms underlying drug-induced changes in D2 autoreceptors. Here, we reported that 5 days of amphetamine (AMPH) self-administration reduced the ability of D2 autoreceptors to inhibit DA release in the NAcc as determined by voltammetry. Using the antibody-capture [(35)S]GTPγS scintillation proximity assay, we demonstrated for the first time that midbrain D2/D3 receptors were preferentially coupled to Gαi2, whereas striatal D2/D3 receptors were coupled equally to Gαi2 and Gαo for signaling. Importantly, AMPH abolished the interaction between Gαi2 and D2/D3 receptors in the midbrain while leaving striatal D2/D3 receptors unchanged. The disruption of the coupling between D2/D3 receptors and Gαi2 by AMPH is at least partially explained by the enhanced RGS2 (regulator of G-protein signaling 2) activity resulting from an increased RGS2 trafficking to the membrane. AMPH had no effects on the midbrain expression and trafficking of other RGS proteins such as RGS4 and RGS8. Our data suggest that midbrain D2/D3 receptors are more susceptible to AMPH-induced alterations. Reduced D2 autoreceptor function could lead to enhanced DA signaling and ultimately addiction-related behavior. RGS2 may be a potential non-dopaminergic target for pharmacological intervention of dysfunctional DA transmission and drug addiction.

Figures

Figure 1
Figure 1
Amphetamine (AMPH) self-administration results in an escalation of rate of intake over sessions. Rat received AMPH (0.187 mg/kg/infusion) on a fixed-ratio one schedule of reinforcement for five consecutive days with a maximum of 40 infusions per day. Session 1 is counted as the first session that occurs following acquisition. (a) Representative operant behavior from a rat undergoing AMPH self-administration. Tick marks represent AMPH infusions delivered. (b) The rate of AMPH intake (infusion/hr/day) from AMPH self-administering rats was averaged. Rats escalate their rate of intake over days (N=6). Data are reported as mean±SEM.
Figure 2
Figure 2
Amphetamine (AMPH) self-administration results in increased evoked dopamine (DA) release and subsensitive D2-like autoreceptors. (a) Representative DA traces showing evoked DA release over time as measured by voltammetry. (b) Evoked DA release was enhanced following AMPH self-administration in the nucleus accumbens core compared with controls. (c) The maximal rate of DA uptake (Vmax) via the DA transporter (DAT) remains unchanged following AMPH self-administration. (d) The ability of D2/D3 autoreceptors in inhibition of evoked DA release from the nucleus accumbens core was dose-dependently decreased following AMPH self-administration as compared with controls. Data are expressed as mean±SEM (N=6–7). *P<0.05; **P<0.01 vs controls.
Figure 3
Figure 3
AMPH self-administration reduced phosphorylation of TH (pTH) in the midbrain. (a) Representative western blots of midbrain samples from a control and an AMPH self-administration rat. Samples were blotted for pTH at Ser 31 and Ser 40, and total TH. (b) Summary of the expression of the pTH at Ser 31 and 40. pTH was normalized to its own total TH and was calculated as a ratio relative to the value of control animals. Data are expressed as mean±SEM (N=5–6), *P<0.05; **P<0.01 vs controls.
Figure 4
Figure 4
AMPH self-administration had a differential effect on D2/D3 receptor coupling to Gαi/o proteins in the midbrain and striatum. (a) Midbrain or (b) striatal membranes from control and amphetamine-treated rats were assayed for individual G-protein activation by quinpirole (10 μM) using a antibody-capture [35S]GTPγS scintillation proximity assay as described in the Materials and Methods. Data were reported as percent increase over basal (no quinpirole) (N=4–6). In the midbrain, D2/D3 receptors were primarily coupled to Gαi2, which was abolished by AMPH self-administration (a). In contrast to the midbrain, striatal D2/D3 receptors were primarily coupled to Gα2 and Gαo, and AMPH self-administration did not have any effects on D2/D3 receptors and Gαi/o proteins (b). Data are expressed as mean±SEM. *P<0.05, **P<0.01 compared with the basal level.
Figure 5
Figure 5
Differential effects of AMPH self-administration on the expression of RGS genes and proteins. (a) AMPH self-administration induced a significant increase in levels of RGS2 and RGS4 mRNA in the midbrain when compared with the control treatment (N=5). However, AMPH self-administration did not alter the mRNA expression of RGS8. Data were normalized by the average mRNA expression of actin and GAPDH. (b) AMPH self-administration did not cause any changes in the total protein expression of RGS2, RGS4, and RGS8 proteins in the midbrain. Data were normalized by actin (N=5). (c) AMPH self-administration significantly reduced RGS2 protein expression in the striatum when compared with controls (N=6). Data are expressed as mean±SEM. *P<0.05, **P<0.01 vs controls.
Figure 6
Figure 6
AMPH self-administration induced differential trafficking of RGS2, RGS4, and RGS8 proteins in the midbrain. Top panels: representative western blots of the midbrain RGS2, RGS4, and RGS8 protein expression in the membrane and the cytosol of a control (c) and an AMPH (a) self-administering rat. Bottom panels: a summary of the membrane and the cytosol expression of RGS2 (a), RGS4 (b), and RGS8 (c) in the midbrain. AMPH self-administration resulted in an increase in the membrane insertion and a decrease in the cytosolic translocation of RGS2 proteins without any effects on RGS4 or RGS8 protein trafficking (N=4–5). Data were normalized by actin, and expressed as mean±SEM. *P<0.05, **P<0.01 vs controls.

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