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Supplemental Materials and Methods SNc dopamine neurons were identified using conventional criteria (Ford et al, 2006; Grace and Onn, 1989): 1) Anatomical location and large soma diameter; 2) Presence of a significant hyperpolarization–activated membrane rectification (Ih current) when the membrane potential was hyperpolarized. Only dopamine neurons in the SNc display the Ih current. In some experiments, post-hoc immunohistochemical staining for the presence of tyrosine hydroxylase (TH) was used to verify recorded neurons as dopamine neurons. Electrophysiological recordings Briefly, rats were deeply anesthetized with isoflurane and brains were rapidly removed and placed in ice cold "cutting buffer" containing (in mM): 75 NaCl, 2.5 KCl, 0.1 CaCl2, 6 MgSO4, 1.2 NaH2PO4, 25 NaHCO3, 2.5 D-Dextrose, 50 Sucrose. Midbrain horizontal slices (~230 mm) were cut in 95% O2 and 5% CO2 oxygenated cutting buffer. Slices were incubated in warm (35 °C) 95% O2 and 5% CO2 oxygenated artificial cerebrospinal fluid (aCSF) containing kynurenic acid (500 nM) for at least 40 min and maintained at room temperature afterward until transfer to a recording chamber. The aCSF contained (in mM): 126 NaCl, 2.5 KCl, 2.4 CaCl2, 1.2 MgCl2, 1.2 NaH2PO4, 21.4 NaHCO3, 11.1 D-Dextrose, pH 7.4, and the osmolarity was adjusted to 300-310 mOsm. Whole cell patch clamp recordings were made from visually identified SNc DA neurons under an Olympus BX51W1 upright microscope (Olympus, Lake Success, NY) equipped with infrared optics. Patch pipettes were pulled from borosilicate glass (1.5 mm diameter; WPI, Sarasota, FL) on a two-stage puller (PP83, Narishige, Tokyo, Japan). Pipettes had a resistance of 2-4 Mohm when filled with the intracellular solution 1 containing (in mM): 130 CsCl, 5.4 KCl, 0.1 CaCl2, 2MgCl2, 10 HEPES, 1.1 EGTA, 30 DDextrose, 4 Mg-ATP, 1 Na-GTP; pH was adjusted to 7.2-7.3, and the osmolarity was adjusted to 280-290 mOsm. Whole cell series resistance was compensated ~80%. Oxygenated aCSF at 32 °C was used for recording and extracellular solution exchange was achieved by height controlled perfusion system with speed ~3-4 ml/min. Whole cell currents and evoked excitatory synaptic currents (eEPSCs) were recorded from SNc DA neurons at a holding potential of -70 mV unless otherwise noted. Currents were collected at 2 kHz and digitized at 5 kHz using either an Axopatch 700A or 200B amplifiers controlled by Axograph Data Acquisition software. NMDAR EPSCs were evoked in nominally Mg2+-free aCSF solution containing 10 µM NBQX and 10 µM glycine with a bipolar stimulating electrode placed ~200-300 mm distally from the recorded cell. Stimulation pulses (0.02 ms) were delivered at 0.05 Hz. During each experiment, a voltage step of -10 mV from the holding potential was applied periodically to monitor cell capacitance and access resistance. Recordings in which access resistance or capacitance changed by >15% during the experiment were excluded from data analysis. To avoid activation of DA receptors and alpha-adrenergic receptors caused by increases in extracellular DA release, the following inhibitors were included in the aCSF recording solution: DA- -D2: eti alpha-adrenergic receptors: prazosin 1 µM. Bicuculline (10 µM) and strychnine (1 µM) were also included in the bath during recordings to eliminate GABAA and glycine receptor activation. For paired-pulse ratio measurements, two stimuli were applied at an interval of 50 ms. The paired–pulse ratio was calculated by dividing the amplitude of EPSC evoked by the second stimulus by the amplitude of the EPSC evoked by the first 2 stimulus (P2/P1). One neuron was recorded per slice and two slices were typically recorded per rat. Each set of experiments was repeated using at least 3 distinct rats with no more than 2 cells from a single rat included in a specific dataset. Surface biotinylation: Cell surface biotinylation was performed in ice cold buffer (in mM: 2 CaCl2, 150 NaCl, and 10 triethanolamine, pH 7.5) with 2 mg/ml sulfosuccinimidyl 2(biotinamido) methyl-1,3-dithiopropionate (sulfo-NHS-SS-biotin; Pierce, Rockford, IL) for 20 minutes at 4C. Non-bound biotin was quenched with a 20 minute wash in 100 mM glycine in PBS and cells were lysed in buffer comprised of 1% Triton X-100, 150 mM NaCl, 5 mM EDTA, and 50 mM Tris, pH 7.5, containing a protease inhibitor mixture (1; Roche Molecular Biochemicals, Indianapolis, IN USA). Biotinylated proteins were isolated with a 2 hour incubation with Ultralink immobilized NeutrAvidin beads (Pierce) washed first with lysis buffer, then a high-salt wash buffer (0.1% Triton X-100, 500 mM NaCl, 5 mM EDTA, and 50 mM Tris, pH 7.5) and finally 50 mM Tris, pH 7.5. Chemicals: AMPH and MA (Sigma, St. Louis, MO USA), DL-2-amino-5phosphonopentanoic acid (AP5), 2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo[f]quinoxaline2,3-dione (NBQX), MK-801, bicuculline, kynurenic acid (KA), ifenprodil, RO 04-5595, RO 25-9681, UBP141, R-CPP (Abcam, Cambridge, MA USA), SCH23390, eticlopride, strychnine (Sigma), prazosin and TCN 201 (Tocris Bioscience, Bristol UK) were dissolved according to the manufacturer's instructions and made in stock solution kept in -20°C then diluted to final concentration using aCSF on the day of the experiment. 3 Behavioral Study Adult (73-118 day old) male and female MA low drinking (MALDR) mice from the MA Abuse Research Center Animal Core within the VA Portland Health Care System animal facility were used for this study. These mice were originally produced by selective breeding to study genetic factors that impact MA intake (Shabani et al, 2011; Wheeler et al, 2009). They were chosen for the current study because their genetic background is a mixture from the C57BL/6J and DBA/2J inbred strains, and this mixed genetic background improves generalizability of the results, compared to use of a single inbred strain. In addition, they are known to exhibit locomotor stimulation in response to MA (Shabani et al, 2011). Mice were weaned at 21 ± 1d of age and maintained in samesex groups in acrylic plastic shoebox style cages (28.5 × 17.5 × 12 cm) with wire tops and Bed-O-CobTM bedding (Animal Specialties Inc., Hubbard, OR, USA). They had free access to tap water and rodent block chow (Purina 5001TM, 4.5% fat content; Animal Specialties Inc., Hubbard, OR, USA), except during activity testing, and room temperature was 21 ± 1°C on a L:D cycle of 12:12 h, with lights on at 0600 h, with testing occurred between 1000 and 1445 h (n = 4-9 per sex per RO group per MA group). Methamphetamine (MA) was utilized in the behavioral study to increase relevance to human drug use, since MA is the more commonly abused drug. However, amphetamine is a metabolite of MA (Hendrickson et al, 2006) and could have been involved in the locomotor stimulant effects seen here. The range of doses that produce locomotor stimulation in mice is similar for amphetamine and MA (Phillips et al, 2008) and they share common mechanisms of transmitter release (Fleckenstein et al, 2007). 4 Locomotor activity testing was performed on 3 consecutive days to allow for habituation to handling and injection (Day 1), collection of baseline activity data (Day 2) and collection of data after drug treatment (Day 3). RO 04-5595 HCl (Abcam) and (+)MA HCl (Sigma) were dissolved in sterile physiological saline on the morning of the study. Activity tests were 60 min in duration and data were collected in 5-min periods in automated activity monitors (Accuscan; 20 x 40 x 40 cm, H x W x L), described previously (Gubner and Phillips, 2015). On each test day, mice were moved to the test room 45 min prior to handling to acclimate to the new environment. For testing, mice were weighed, placed in bedding-lined holding cages within 10 min of treatment and given 2 IP injections, spaced 30 min apart. After the second injection, mice were placed into the center of activity chambers for automated recording of activity. On Days 1 and 2, mice received two injections of saline. On Day 3, saline or one of 3 doses of RO 045595 HCl (RO; 5, 10 or 20 mg/kg) was administered 30 min prior to saline or 2 mg/kg MA. This dose of MA was chosen as a dose with stimulant effects in MALDR mice (Shabani et al, 2011). Doses of RO were selected by pilot testing for a potential effect on MA-induced stimulation in the absence of a significant effect in saline-treated mice. Data Analysis: Data are reported as mean ± SEM, and were analyzed by repeated measures ANOVA, with time as the repeated measure, and sex, RO dose, and MA dose as independent variables. The main dependent variable was Day 3 drug activity corrected for Day 2 baseline activity by subtraction (D3–D2); this provided a Drug Response Score that controlled for differences in baseline activity level among animals. Although there were time-dependent effects, interpretation with regard to treatment effects was largely the same when data were accumulated for the 60 min time period or 5 examined for smaller blocks of time when drug effects were most apparent. There were no significant effects of sex, so data were collapsed on this factor and simple effects analyses were used to further examine the significant interaction of RO dose and MA dose, followed by Newman Keuls mean comparisons, when appropriate. Supplemental Results AMPH-mediated potentiation of NMDA EPSCs is dependent on DAT All recordings were done in a cocktail of inhibitors of dopamine receptors, so AMPH-induced increases in extracellular dopamine and activation of dopamine receptors did not play a role in AMPH potentiation of NMDAR EPSCs. In order to test whether AMPH activation of DAT was required for potentiation, the DAT inhibitor GBR12909 (1 µM) was superfused prior to AMPH and was observed to completely block potentiation of NMDAR EPSCs by AMPH (GBR12909: -184 ± 18 compared to GBR12909 + AMPH: -200 ± 20 pA, Fig. S1A,B; see potentiation by AMPH alone in Fig. S1C). This result suggests that AMPH modulates excitatory synaptic transmission via its direct substrate activity at the DAT. We also tested whether continued activation of the DAT by AMPH was required to maintain potentiated NMDAR EPSCs. The potentiation by AMPH was slow to develop (>10 min post-superfusion) and lasted as long as the whole-cell recording. In experiments where we attempted to wash out AMPH (> than 30 min), we saw no degradation in the potentiation (data not shown). However, given that AMPH is a lipophilic molecule, it is unlikely that all of the AMPH washes out during the time course of the whole-cell recordings. In a subset of experiments, AMPH was superfused to elicit 6 potentiated EPSCs, and then GBR12909 (1 µM) was superfused to determine if blockade of DAT would reduce NMDAR EPSCs. We observed no reversal of AMPH’s effects in the presence of GBR12909 (-291 ± 32 pA) compared to AMPH alone (-261 ± 8 pA; n = 3; Repeated measures One way ANOVA; F(2,4) = 9.68, p = 0.03; Fig. S1C) suggesting that the DAT is required for transporting AMPH into the cell to initiate the potentiation, but is not required for continued expression of potentiated NMDAR EPSCs. AMPH and MA-induced potentiation of NMDAR EPSCs are reduced at +40 mV EAAT3 transport is voltage dependent and is dramatically reduced at +40 mV (Wadiche et al, 1995). In experiments using rats (Fig. S2A,B), the AMPH-mediated potentiation of NMDAR EPSCs measured 40 ms after the peak at +40 mV was 21 ± 7%, smaller than the potentiation measured at -70 mV in 0 Mg2+ extracellular solution (46 ± 8%, t(22) = 2.15, p = 0.04). The effect was not as dramatic with MA-mediated potentiation in rats (46 ± 10% at +40 mV compared to 58 ± 13% at -70 mV, t(21) = 0.70, p = 0.49). In recordings from mice (Fig. S2C,D), we observed many cells in which NMDAR EPSCs measured at +40mV were not potentiated by either AMPH or MA. The AMPH-mediated potentiation of NMDAR EPSCs measured at +40 mV was 8 ± 6%, substantially smaller than the potentiation measured at -70 mV in 0 Mg2+ extracellular solution (46 ± 13%, t(14) = 3.02, p = 0.009). The MA-induced potentiation of NMDAR EPSCs at +40 mV was also smaller (6 ± 2%) compared to the increase measured at -70 mV in 0 Mg2+ extracellular solution (118 ± 23%, t(5.1) = 4.88, p = 0.004 with Welch’s correction). The lack of AMPH and MA effects at +40 mV in mice may be due to differences in space-clamp control of recordings in rats and mice (Courtney et al, 2012). 7 NMDAR NR2A/B receptor subunits are required for the potentiation of NMDA synaptic transmission by AMPH The effects of additional NMDAR subunit-specific inhibitors were used to determine the subunit composition of NMDARs that are stimulated under baseline conditions. TCN 201 is a selective inhibitor of GluN2A subunits (Hansen et al, 2014; Izumi and Zorumski, 2015). There was no effect of TCN 201 at either 3 µM (Repeated Measures One way ANOVA, F(2,10) = 1.49, p = 0.27) or 10 µM (Repeated Measures One way ANOVA, F(2,8) = 0.91, p = 0.44) on basal NMDAR EPSCs but TCN 201 appeared to block AMPH potentiation of NMDAR EPSCs at both concentrations (Fig. S3A,B). R-CPP is a selective inhibitor for NR2A at low concentrations (Ki = 40 nM) but inhibits all NR2 subunits at higher concentrations (Ki = 300 nM for NR2B, 600 nM for NR2C and 2 µM for NR2D). The NR2A-selective concentration of R-CPP (50 nM) did not inhibit basal NMDAR EPSCs but also had a trend toward an increase in the absence of AMPH (Repeated Measures One way ANOVA, F(2,6) = 5.04, p = 0.052; Fig. S3C,D). However, the effect of AMPH was blocked (or occluded) by this concentration of R-CPP (n = 4). These results suggested that NR2A subunits contribute to NMDARs activated in the presence of AMPH. The trend toward an increase in NMDAR EPSCs in the presence of TCN 201 and R-CPP (50 nM) suggest the presence of triheteromers. As expected, a high concentration of R-CPP (2 µM) significantly decreased the amplitudes of basal NMDAR EPSCs (One way ANOVA, F(2,8) = 57.35, p < 0.0001; Fig. S3E,F). In some but not all SNc neurons, R-CPP (2 µM) abolished the NMDAR-mediated synaptic currents. 8 In all recordings tested, the potentiation by AMPH was blocked in the presence of RCPP (2 µM). AMPH does not affect kinetics of NMDAR EPSCs A rise in extracellular glutamate could increase spill-over from the synapse and activate extrasynaptic glutamate receptors (Chen and Diamond, 2002; Harney et al, 2008; Scimemi et al, 2009). The rise time and decay kinetics of EPSCs were compared in the absence and presence of AMPH (Fig. S4). Weighted decay time constants were calculated from double exponential fits with the equation w = [If/(If + Is)] X f + [Is/(If + Is)] X s, where If and Is are the amplitudes of the fast and slow decay components and f and s are the decay time constants (Vicini et al, 1998). First, there was no effect of AMPH on either the 10-90% rise time (Paired t-test, t(4) = 0.88, p = 0.21) or decay kinetics of AMPAR-mediated EPSCs (Paired t-test, t(5) = 1.67, p = 0.16; Fig. S4A). Similarly, AMPH did not alter 10-90% rise times of NMDAR EPSCs (4.2 ± 0.7 compared to 4.5 ± 0.6 ms (Paired t-test, t(9) = 0.59, p = 0.57). NMDAR-mediated EPSC decay time constants showed a trend toward an increase in the presence of AMPH (72 ± 21 ms compared to 47 ± 12 ms, Paired t-test, t(13) = 1.99, p = 0.07, Fig. S4B). 9 Figure S1. AMPH potentiation of postsynaptic NMDARs is DAT dependent. A. Representative averaged evoked NMDAR EPSCs (baseline), following superfusion of the DAT inhibitor, GBR12909 (1 µM) or GBR12909 with AMPH (10 µM). B. Summary data of NMDAR EPSC amplitudes in each condition. The AMPH potentiation (shown in C) was blocked by GBR12909 (n = 5). C. Summary of results when AMPH was applied prior to GBR12909 (Repeated measures One-way ANOVA, F(2,4) = 9.68, p = 0.03, *Holm-Sidak multiple comparison, p < 0.05). 10 Figure S2. The voltage-dependence of EAAT3 activity reduces the effects of AMPH and MA at +40 mV. A and B, Summary data comparing the effects of AMPH (A) and MA (B) on NMDAR EPSCs measured at -70 mV in 0 Mg2+ extracellular solution and +40 mV in normal extracellular solution in rats. C and D, Summary data comparing the effects of AMPH (C) and MA (D) on NMDAR EPSCs measured at -70 mV in 0 Mg2+ extracellular solution and +40 mV in normal extracellular solution in C57 mice. * t-test, p<0.05 11 Figure S3. Basal NMDAR EPSCs consist primarily of NR2C subunit-containing receptors while AMPH potentiation is due to recruitment of NR2A/NR2B subunitcontaining receptors. A. Time course of a single experiment testing the selective GluN2A inhibitor TCN 201 (10 µM). There was no inhibition of basal NMDAR EPSCs at this concentration but the effect of AMPH was blocked. B. Summary data of NMDAR EPSC amplitudes for experiments with TCN 201 (3 µM, n = 6; 10 µM, n = 5 combined). C. Time course of a single experiment testing R-CPP (50 nM), a concentration selective for NR2A subunits. There was no inhibition of basal NMDAR EPSCs at this 12 concentration but the effect of AMPH was blocked. D. Summary data of NMDAR EPSC amplitudes for experiments with R-CPP (50 nM, n = 4). E. Time course of a single experiment testing R-CPP (2 µM), a nonselective concentration of R-CPP at NMDAR NR2 subunits. In this cell, R-CPP significantly decreased basal NMDAR-mediated EPSCs. AMPH potentiation was blocked in all cells at this concentration of R-CPP. F. Summary data of NMDAR EPSC amplitudes for experiments with R-CPP (2 µM; Repeated measures One-way ANOVA, F(2,4) = 57.35, p = 0.001, *Bonferroni multiple comparisons, p < 0.05). 13 Figure S4. AMPH effects on NMDAR EPSC rise time and decay kinetics. Rise time and weighted decay time calculated from exponential fits to AMPAR (A) and NMDAR (B) EPSCs measured at -70 mV. 14 References Chen S, Diamond JS (2002). Synaptically released glutamate activates extrasynaptic NMDA receptors on cells in the ganglion cell layer of rat retina. J Neurosci 22(6): 21652173. Courtney NA, Mamaligas AA, Ford CP (2012). Species differences in somatodendritic dopamine transmission determine D2-autoreceptor-mediated inhibition of ventral tegmental area neuron firing. J Neurosci 32(39): 13520-13528. Fleckenstein AE, Volz TJ, Riddle EL, Gibb JW, Hanson GR (2007). New insights into the mechanism of action of amphetamines. 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