Introduction

Exposure to psychostimulant drugs like amphetamine can cause short and long-term changes to the dopamine (DA) system. Amphetamine is known to powerfully increase extracellular DA and can rapidly induce receptor internalization^1,2. This initial adaptation mechanism in response to drug-induced DA release that can have lasting effects of several hours up to days. Reductions in dopamine D₂/D₃ receptor (D₂/D₃R) availability, measured using positron emission tomography (PET), has been a robust finding in stimulant drug users. Yet, microdialysis and functional imaging studies have also reported discrepancies between the expected timeline of DA release/uptake and paradoxically prolonged reductions in PET signal^3,4. In vitro data have suggested that the rate of receptor recycling can vary between the D₁ and the D₂R subtypes⁵, yet, little is known about the functional effects in vivo. In this study, we investigated the functional consequences of differential D₁ and D₂R trafficking mechanisms following stimulant exposure by imaging with combined PET/fMRI during repeated amphetamine injections in non-human primates.

Methods

Simultaneous PET/fMRI was acquired in three anesthetized rhesus macaques in a total of 20 imaging sessions: The D₂/D₃R PET radiotracer [¹¹C]raclopride was administered as a bolus+infusion, while gradient-echo EPI was acquired continuously with a total scan duration of 2h. Ferumoxytol was injected before the start of fMRI acquisitions as a contrast agent. In 14 sessions, a first (0h) amphetamine dose (0.6 mg/kg) was given intravenously at 40 min after radiotracer administration as a within-scan challenge, followed by a second amphetamine injection after 3h or 24h using the same PET/fMRI acquisitions (Fig. 1). In an analogous set of 6 imaging sessions, SCH23390 was administered as a bolus+infusion (0.1 mg/kg + 0.09 mg/kg/h) to block D₁R prior to radiotracer injection. FMRI data were analyzed with a general linear model and converted to relative changes in cerebral blood volume (CBV). Binding potential (BP_ND) and receptor occupancy were quantified using a modified simplified reference tissue model with a time-dependent binding term^6,7.

Results

After each amphetamine injection, a reduction in striatal [¹¹C]raclopride-PET binding was observed, driven by amphetamine-induced DA release (Fig. 2). Amphetamine-induced CBV changes demonstrated both a positive and negative CBV component that were modulated and interestingly shifted in sign with repeated injections, as seen on the voxelwise maps (Fig. 2). For the first 0h amphetamine injection, we observed a reduction in [¹¹C]raclopride BP_ND, equivalent to a peak occupancy of 27.3% [19.1; 35.1] (mean, [95% confidence interval]), together with a short-lived decrease in CBV with a negative peak at -11.2% [-13.4; -9.0] in the putamen (Fig. 3A,D, black lines). The repeated amphetamine administration after 3h caused a further reduction in BP_ND, i.e. an increase in D₂/D₃R occupancy with combined peak occupancy of 46.6% [37.2; 94.6] (Fig. 3B). In contrast to the first drug injection, the CBV response at 3h showed an entirely different functional response: a long-lasting increase in CBV with a peak change of 14.2% [-5.4; 33.9] (Fig. 3E, black lines). At 24h, D₂/D₃R occupancy had returned to baseline, but showed reduced acute amphetamine-induced occupancy of 13.9% [2.2; 25.6]. The repeated amphetamine administration at 24h caused a biphasic CBV response, consisting of a negative component (-7.3% [-14.8; 0.2]), similar to the 0h response, but exhibiting a longer-lasting positive component (4.1% [-1.3; 9.6]), as seen with the amphetamine injection at 3h (Fig. 3C,F, Fig. 4A-C).
Administration of SCH23390 neither altered the overall shape of the amphetamine-induced changes in CBV at 0h (Fig. 3D, pink lines), nor appreciably affected BP_ND. Most interestingly, the long-lasting positive CBV response at 3h was reduced to a short-lived positive response (Fig. 3E, pink lines), demonstrating the D₁-specificity of the functional response. At 24h, the positive, excitatory component of the CBV response was also attenuated, further providing evidence of the D₁ component of the response. Overall, CBV responses behaved similarly in regions of the basal ganglia (putamen, caudate, nucleus accumbens). The thalamus exhibited a slightly different CBV response, with a biphasic CBV response at both 0h and 24h, and a more moderate positive CBV signal at repeated amphetamine injections that was not eliminated by SCH23390 blocking at 24h (Fig. 4).

Discussion

The results from this combined PET/fMRI study demonstrate (i) persistent D₂/D₃ receptor internalization after even just one amphetamine exposure at 0h that lasts for up to 24h, (ii) continued DA release with repeated injections and (iii) differences in functional activation between D₁ and D₂/D₃R within 3h of repeated amphetamine exposure. The elimination of the dominant positive CBV response at 3h after D₁ receptor blocking demonstrates the D₁-specificity of the functional response. Our data suggest that excitatory vs. inhibitory DA receptor subtypes have varying time constants for internalization and recycling, with inhibitory D₂/D₃R taking a longer time to recycle/externalize compared to D₁ receptors after amphetamine exposure. We present these novel results on the modulation of both D₁ vs. D₂R activity due to repeated amphetamine in a translational in vivo setting. Our data uniquely show the adaptation of receptors over time in vivo, thereby providing insight into adaptive changes beyond the D₂/D₃ receptors, and the potential role of D₁R that may be of relevance for understanding the molecular mechanisms of stimulant use and abuse.

Acknowledgements

This work was supported by NIH grants K99DA043629, R00DA043629, P41EB015896, S10RR026666, S10RR022976, S10RR019933 and S10RR017208.