Amphetamine and Methamphetamine Increase NMDAR-GluN2B Synaptic Currents in Midbrain Dopamine Neurons

Neuropsychopharmacology (2017) 42, 1539–1547 © 2017 American College of Neuropsychopharmacology. All rights reserved 0893-133X/17 www.neuropsychopharmacology.org Amphetamine and Methamphetamine Increase NMDAR-GluN2B Synaptic Currents in Midbrain Dopamine Neurons Ming-Hua Li1, Suzanne M Underhill2, Cheryl Reed3, Tamara J Phillips3,4, Susan G Amara2 and Susan L Ingram*,1 1 Department of Neurological Surgery, Oregon Health & Science University, Portland, OR, USA; 2National Institute of Mental Health, National Institutes of Health, Laboratory of Molecular and Cellular Neurobiology, Bethesda, MD, USA; 3Department of Behavioral Neuroscience, Oregon Health & Science University, Portland, OR, USA; 4VA Portland Health Care System, Portland, OR, USA The psychostimulants amphetamine (AMPH) and methamphetamine (MA) are widely abused illicit drugs. Here we show that both psychostimulants acutely increase NMDA receptor (NMDAR)-mediated synaptic currents and decrease AMPA receptor (AMPAR)/ NMDAR ratios in midbrain dopamine neurons. The potentiation depends on the transport of AMPH into the cell by the dopamine transporter. NMDAR-GluN2B receptor inhibitors, ifenprodil, RO 25-6981, and RO 04-5595, inhibit the potentiation without affecting basal-evoked NMDA currents, indicating that NMDAR-GluN2B receptors are activated by AMPH. A selective peptide inhibitor of AMPHdependent trafficking of the neuronal excitatory amino acid transporter 3 (EAAT3) blocks potentiation, suggesting that EAAT3 internalization increases extracellular glutamate concentrations and activates GluN2B-containing NMDARs. Experiments with the usedependent NMDAR blocker, MK-801, indicate that potentiated NMDARs reside on the plasma membrane and are not inserted de novo. In behavioral studies, GluN2B inhibitors reduce MA-mediated locomotor activity, without affecting basal activity. These results reveal an important interaction between dopamine and glutamatergic signaling in midbrain dopamine neurons in response to acute administration of psychostimulants. Neuropsychopharmacology (2017) 42, 1539–1547; doi:10.1038/npp.2016.278; published online 18 January 2017 INTRODUCTION Repeated use of the psychostimulants amphetamine (AMPH) and methamphetamine (MA) leads to long-term adaptations in the central nervous system, including locomotor sensitization (Karler et al, 1989; Kelly et al, 2008) that can be observed following a single injection of AMPH (Vanderschuren et al, 1999). The cellular mechanisms underlying sensitization are not well understood, but AMPH or MA administration is associated with increased dopamine and glutamate release in midbrain areas (Lominac et al, 2012; Vanderschuren et al, 1999; Wolf et al, 2000; Zhang et al, 2001), suggesting an intricate interplay between these two neurotransmitter systems in midbrain circuits. Indeed, locomotor sensitization can be blocked with NMDA receptor (NMDAR) antagonists (Karler et al, 1989; Yap et al, 2005), implying that NMDARs have an important role in synaptic plasticity induced by psychostimulants. *Correspondence: Dr SL Ingram, Department of Neurological Surgery, Oregon Health & Science University (OHSU), 3181 SW Sam Jackson Park Road, Portland, OR 97239, USA, Tel: 503 494 1220, Fax: 503 494 2664, E-mail: Received 29 August 2016; revised 29 November 2016; accepted 11 December 2016; accepted article preview online 15 December 2016 AMPH and MA have multiple actions on dopamine neurons; they are substrates for the dopamine transporter (DAT) and vesicular monoamine transporter (Sulzer, 2011) and induce trafficking of DAT (Johnson et al, 2005; Kahlig et al, 2006; Saunders et al, 2000). We recently showed that AMPH also induces trafficking of the neuronal excitatory amino acid transporter 3 (EAAT3) from the plasma membrane of dopamine neurons (Underhill et al, 2014). This family of glutamate transporters includes five isoforms that regulate the temporal and spatial influence of glutamate at the synapse and protect neurons from glutamate excitotoxicity. In general, EAAT isoforms 1 and 2 are found predominantly in astrocytes, EAAT3 in neurons, EAAT4 in cerebellar Purkinje cells, and EAAT5 expression is restricted to the retina (Danbolt, 2001). EAAT3 trafficking increased NMDAR synaptic currents in mouse dopamine neurons localized to both the substantia nigra pars compacta (SNc) and ventral tegmental area (Underhill et al, 2014). Here, we further explore the effects of AMPH and MA on glutamatergic synaptic currents in SNc dopamine neurons. We show that AMPH superfusion potentiates NMDAR-mediated synaptic currents and that the potentiation is dependent on the activation of NMDARs containing GluN2B subunits. Further, activation of NMDARGluN2B subunits contributes to locomotor stimulation induced by acute administration of MA. AMPH increases NMDAR-GluN2B EPSCs M-H Li et al 1540 Figure 1 Amphetamine (AMPH) increases NMDA receptor (NMDAR) excitatory postsynaptic currents (EPSCs) in substantia nigra pars compacta (SNc) dopamine neurons. (a) Isolated NMDAR responses were significantly potentiated by AMPH (n = 14), but AMPA receptor (AMPAR) EPSCs were unaffected (n = 8). (b) To determine AMPAR/NMDAR ratios, evoked AMPAR and NMDAR EPSC amplitudes were measured at +40 mV at the peak and 40 ms later, respectively. AMPAR/NMDAR ratios were significantly decreased after superfusion of AMPH (10 μM) for 20 min (n = 7). (c) The potentiation by AMPH was observed in cells expressing a hyperpolarization-activated current (Ih). (d) No potentiation occurred in Ih negative SNc neurons. *po0.05. MATERIALS AND METHODS Approvals All procedures were performed in strict accordance with the Guide for the Care and Use of Laboratory Animals as adopted and promulgated by the National Institutes of Health and approved by the Institutional Animal Care and Use Committees of Oregon Health & Science University and the VA Portland Health Care System. In Vitro Studies Rats. Postnatal days 25–45 male Sprague–Dawley rats were used for all electrophysiology experiments. Midbrain horizontal slices were prepared and the electrophysiological and biochemical experiments performed as described previously (Underhill et al, 2014). Detailed methods and chemicals used are described in Supplementary Materials and Methods. Data analysis. All data are reported as mean ± SEM. Student’s t-test and analysis of variance (ANOVA) were used where appropriate. The criterion for significance was set at po0.05. Behavioral Study Adult (73–118 days old) male and female MA low drinking mice from the MA Abuse Research Center Animal Core within the VA Portland Health Care System animal facility were used for this study. Locomotor activity testing was performed on 3 consecutive days to allow for habituation to Neuropsychopharmacology handling and injection (Day 1), collection of baseline activity data (Day 2), and collection of data after drug treatment (Day 3). RO 04-5595 (RO) HCl (Abcam, Cambridge, MA) and (+)MA HCl (Sigma, St Loui (...truncated)