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. Author manuscript; available in PMC: 2021 Jul 1.
Published in final edited form as: Addict Biol. 2019 May 6;25(4):e12763. doi: 10.1111/adb.12763

A history of ethanol drinking increases locomotor stimulation and blunts enhancement of dendritic dopamine transmission by methamphetamine

Christopher W Tschumi 1,2, Anna W Daszkowski 2, Amanda L Sharpe 3, Marta Trzeciak 1, Michael J Beckstead 1,2
PMCID: PMC6832768  NIHMSID: NIHMS1020125  PMID: 31062485

Abstract

Ethanol and psychostimulant use disorders exhibit comorbidity in humans and cross-sensitization in animal models, but the neurobiological underpinnings of this effect are not well understood. Ethanol acutely increases dopamine neuron excitability, and psychostimulants such as cocaine or methamphetamine increase extracellular dopamine through inhibition of uptake through the dopamine transporter (DAT) and/or vesicular monoamine transporter 2 (VMAT2). Psychostimulants also depress dopamine neuron activity by enhancing dendritic dopamine neurotransmission. Here we show that mice with a previous history of ethanol drinking are more sensitive to the locomotor-stimulating effects of a high dose (5 mg/kg), but not lower doses (1, 3 mg/kg) of methamphetamine or any tested dose of cocaine (3, 10, and 18 mg/kg), compared to water-drinking controls. We next investigated the impact of a history of ethanol drinking, in a separate group of mice, on methamphetamine- or cocaine-induced enhancement of dendritic dopamine transmission using whole-cell voltage clamp electrophysiology in mouse brain slices. Methamphetamine, applied at a concentration (10 µM) that affects both DAT and VMAT2, enhanced D2 receptor-mediated inhibitory postsynaptic currents (D2-IPSCs) in both groups, but this effect was blunted in mice with a history of ethanol drinking. As methamphetamine action at VMAT2 disrupts dopamine neurotransmission, these results may suggest enhanced action of methamphetamine at VMAT2. Furthermore, there were no differences in low-dose methamphetamine or cocaine-induced enhancement of D2-IPSCs, suggesting intact DAT function. Disruption of methamphetamine-induced enhancement of dendritic dopamine transmission would result in decreased inhibition of dopamine neurons, ultimately increasing downstream release and the behavioral effects of methamphetamine.

Keywords: cocaine, dendrodendritic, dopamine, ethanol, methamphetamine, VMAT2

Introduction

Ethanol and psychostimulant use disorders are prevalent in the United States with 16 and 1.5 million affected in 2014, respectively (Hedden et al., 2014). Comorbidity often occurs between these disorders, possibly due to previous ethanol experience (Ford et al. 2009; Salo et al. 2011). Rodent models of drug use exhibit cross-sensitization between the behavioral effects of ethanol and psychostimulants including cocaine, amphetamine, and methamphetamine, however the characteristics of these effects differ between compounds (Manley & Little 1997; Itzhak & Martin 1999; Lessov & Phillips 2003; Abrahao, Quadros & Souza-Formigoni 2009). For example, six days following chronic ethanol administration the locomotor response to amphetamine is enhanced both on the first and in following locomotor sessions, but enhanced sensitivity to cocaine occurs only from the second session onward (Manley & Little, 1997). While behavioral effects have been fairly well-studied, the neuroadaptations that occur following ethanol use that contribute to increased sensitivity to psychostimulants are not well understood.

Psychostimulants such as cocaine and methamphetamine increase extracellular levels of the neurotransmitter dopamine (Di Chiara & Imperato 1988), but do so through different neurobiological processes. Cocaine inhibits the dopamine transporter (DAT), while methamphetamine acts as a substrate both for DAT and the vesicular monoamine transporter VMAT2 (Sulzer, Maidment & Rayport 1993; Branch & Beckstead 2012). The acute effects of psychostimulants on dopamine neuron activity in the substantia nigra pars compacta (SNc) and ventral tegmental area (VTA) are complex. In awake animals, cocaine increases the activity of a subgroup of dopamine neurons (Koulchitsky et al., 2012), but in anesthetized animals as well as ex vivo preparations psychostimulants depress the activity of the majority of dopamine neurons measured (Brodie & Dunwiddie, 1990; Lacey, Mercuri, & North, 1990; Mercuri, Calabresi, & Bernardi, 1992). Interestingly, while cocaine and methamphetamine increase extracellular dopamine, both simultaneously depress dopamine neuron firing activity in part by enhancing dendrodendritic dopamine transmission (Beckstead et al., 2004, Branch & Beckstead, 2012). This form of synaptic inhibition is dependent on D2 autoreceptors and GIRK-type potassium channels, and can be studied in mouse brain slices by measuring D2 receptor-mediated inhibitory postsynaptic currents (D2-IPSCs; Beckstead et al. 2004).

Ethanol does not act directly at DAT but induces neuroadaptations that overlap with the cellular mechanisms by which psychostimulants enhance dopamine neurotransmission. Acutely, ethanol increases extracellular dopamine by increasing dopamine neuron excitability through actions on multiple ionic conductances (Brodie & Appel 1998; Okamoto, Harnett & Morikawa 2006; Beckstead & Phillips 2009). In contrast to acute ethanol which does not alter dopamine uptake (Yim & Gonzales 2000), repeated ethanol exposure followed by abstinence increases dopamine uptake (Karkhanis et al. 2015) and VMAT2 gene expression (Darlington et al. 2014). This suggests long-lasting alterations in dopamine uptake are induced by previous ethanol exposure. Furthermore, cessation of ethanol exposure results in decreased dopamine neuron burst firing, decreased extracellular levels of dopamine, and up or downregulation of D2 receptor function in dopamine neurons (Diana et al. 1996; Karkhanis et al. 2015; Ding et al. 2016). Thus, previous ethanol exposure may disrupt dendritic neurotransmission between dopamine neurons presynaptically by depressing dopamine neuron excitability or postsynaptically by decreasing D2 receptor function. However, it is not clear if these alterations translate to a change in the ability of psychostimulants to enhance dendritic dopamine transmission.

The goal of these studies was to determine the impact of a history of ethanol drinking on sensitivity to the behavioral and cellular effects of methamphetamine. We subjected mice to voluntary ethanol consumption for two weeks using the drinking in the dark paradigm. We show that after cessation of ethanol drinking, mice are more sensitive to the locomotor stimulating effects but less sensitive to the D2-IPSC enhancing effects of methamphetamine in SNc dopamine neurons. In contrast, prior ethanol drinking had no effect on enhancement of D2-IPSCs by cocaine, suggesting that ethanol exposure does not alter dendritic dopamine transmission per se, but modulates enhancement of dopamine neurotransmission in a psychostimulant-specific manner. Decreased methamphetamine-induced D2 autoreceptor activation may result in increased excitability of dopamine neurons, dopamine release, and methamphetamine abuse liability.

Materials and methods

Animals

Male C57BL/6J mice from Jackson Labs (Bar Harbor, ME) were singly housed in polycarbonate boxes with rodent bedding. The vivarium was on a reverse light cycle (12/12-hour light-dark cycle, lights off at 0900 hours). All procedures were approved by the Institutional Animal Care and Use Committees at UT Health San Antonio and the Oklahoma Medical Research Foundation.

Ethanol drinking procedure

For both the locomotor and electrophysiology experiments, mice were individually housed at 6 weeks of age and assigned into ethanol and control groups counterbalancing for body weight. Body weight was measured at least once every two days and remained similar between groups (average of last 5 days of drinking; water, 24.6 ± 0.381 g; ethanol, 24.6 ± 0.270 g, t69 = 0.022, p = 0.98, Fig. S1). One to two weeks after individually housing the mice, the drinking in the dark procedure (Rhodes et al. 2005) was initiated and continued for 14 days. Briefly, once daily beginning three hours into the dark cycle, the home cage water bottles were replaced with 50 mL conical tubes fitted with stainless steel sipper tubes that contained either ethanol (20% w/v) or water (control). After four hours, the ethanol- or water-containing conical tubes were removed and the home cage water bottles were replaced. Control (“dummy”) tubes containing ethanol (20% w/v) or water were placed on empty cages in the same rack daily to estimate non-ingestive fluid loss through dripping, vibrations, and evaporation. Consumption of ethanol and water during the limited access procedure was measured as the difference in bottle weight between the pre- and post- limited-access session, and was corrected for the appropriate “dummy” tube weight lost that day. In cases where the calculated consumption was negative (fluid loss from the dummy tube was greater than from the home cage during limited access) consumption of 0 was reported. Daily intake of ethanol (g/kg) was calculated from the corrected weight of the ethanol consumed and the body weight of the mouse.

Locomotor assessment

One week following the last day of ethanol drinking, some mice underwent locomotor testing. Open field chambers with Plexiglass walls and photobeam arrays (16 inches by 16 inches, Opto-Varimex, Columbus Instruments, Columbus, OH) were used for all tests. A total of five locomotor sessions (one every-other day, one hour each) were performed on each mouse, beginning with a 15-minute habituation period during which the mouse was placed into the chamber followed by three intraperitoneal (i.p.) injections for which the mouse was briefly removed from the open field chamber. The first and last locomotor sessions (locomotor sessions S1 and S2), mice received i.p. saline injections at 15, 30, and 45 minutes into the session. One group of mice was administered i.p. methamphetamine for the second, third, and fourth locomotor sessions (M1, M2, and M3). On these days, the mice received doses of 1, 2, and 2 mg/kg at 15, 30, and 45 minutes into the session, resulting in cumulative doses of 1, 3, and 5 mg/kg methamphetamine over the 60 minute session (Fig. 1 A,C). A second group of mice was administered i.p. cocaine for the second, third, and fourth locomotor sessions (C1, C2, and C3). Thus the mice received doses of 3, 7, and 8 mg/kg at 15, 30, and 45 minutes into the session, resulting in cumulative doses of 3, 10, and 18 mg/kg cocaine over the 60 minutes session (Fig. 1 A,D). Horizontal movement (across the floor of the chamber) was measured using Opto-Varimex AutoTracker software. Net distance traveled was calculated as the distance covered in an individual session subtracted by the distance moved in the first saline session.

Fig. 1. Mice consumed ethanol and were later assessed for methamphetamine or cocaine-induced locomotor activity.

Fig. 1.

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Table depicting timeline of experiments across days (A). Mice were split into two groups and given daily 4-hour access to either ethanol (drinking in the dark; DID Ethanol) or water (DID Water) for 14 days, followed by 7 days in their home cage with ad libitum food and water. All mice then underwent behavioral testing during which they were injected with either saline (S1, S2), methamphetamine (M1, M2, M3), or cocaine (C1, C2, C3). DID ethanol mice consumed ethanol when given access, with stable consumption across days (B). Schematics depicting locomotor sessions (C, D). Hypothetical data depicting the average distance traveled per minute by one mouse during 3-minute bins. Methamphetamine (METH) was administered once every 15 minutes at doses of 1, 2, and 2 mg/kg, resulting in cumulative doses of 1, 3, and 5 mg/kg (C). Cocaine was administered once every 15 minutes at doses of 3, 7, and 8 mg/kg resulting in cumulative doses of 3, 10, and 18 mg/kg (D).

Electrophysiology

Electrophysiological recordings of midbrain dopamine neurons were conducted on a group of mice separate from those that underwent locomotor assessment to avoid confounding effects of previous drug exposure. Briefly, approximately one week (mean; 7.4 ± 0.34 days, range; 5–13 days) after the last day of ethanol self-administration, mice were anesthetized using isoflurane and quickly decapitated. Brains were rapidly extracted and placed in ice-cold carboxygenated (95% O2, 5% CO2) artificial cerebrospinal fluid (aCSF) containing the following (in mM): 126 NaCl, 2.5 KCl, 1.2 MgCl2, 2.4 CaCl2, 1.4 NaH2PO4, 25 NaHCO3, and 11 D-glucose. Kynurenic acid (1 mM) was added to the buffer for the slicing procedure. Horizontal midbrain slices (200 µm) containing the SNC and VTA were obtained using a vibrating microtome (Leica). Slices were incubated for at least 30 min at 34–36°C with carboxygenated aCSF that also contained the NMDA receptor antagonist MK-801 (10–20 µM).

Slices were placed in a recording chamber attached to an upright microscope (Nikon Instruments) and maintained at 34–36°C with aCSF perfused at a rate of 1.5 ml/min. Dopamine neurons were visually identified using gradient contrast optics based on their location in relation to the midline, third cranial nerve, and the medial terminal nucleus of the accessory optic tract (MT). Neurons were further identified physiologically by the presence of one or more of the following characteristics: spontaneous pacemaker firing (1–5 Hz), wide extracellular waveforms (>1.1 ms), or a hyperpolarization-activated current (IH) >100 pA. Recordings are either from SNc neurons located near MT or VTA neurons located at least 100 µm medial to MT. Whole-cell (1.5–2.5 MΩ resistance) and cell-attached (5–8 MΩ resistance) recording pipettes were constructed from thin wall capillaries (World Precision Instruments) with a PC-10 puller (Narishige International) and filled with an internal solution consisting of the following (in mM): 115 K-methylsulfate, 20 NaCl, 1.5 MgCl2, 10 HEPES, 2 ATP, 0.4 GTP, and 10 BAPTA, 269–274 mOsm for whole-cell or 135 Na-HEPES and 20 NaCl, 290 mOsm for firing, pH 7.35–7.40.

Dopamine D2 autoreceptor inhibitory postsynaptic currents (D2-IPSCs) were evoked during voltage-clamp recordings (−55 mV) using electrical stimulation in the presence of the following receptor antagonists: picrotoxin (100 µM, GABAA), CGP 55845 (100 nM, GABAB), DNQX (10 µM, AMPA), and hexamethonium (100 µM, nAChR). A bipolar stimulating electrode was inserted into the slice 100–200 µm caudal to the cell, and D2-IPSCs were evoked with a train of 5 stimulations (0.5 ms) applied at 50 Hz once every 50 seconds.

Drugs

Methamphetamine hydrochloride and cocaine hydrochloride were gifts from the National Institute on Drug Abuse Drug Supply Program (Bethesda, MD). Ethanol, kynurenic acid, MK-801, DNQX, picrotoxin, and hexamethonium were purchased from Sigma-Aldrich. CGP55845 was from Tocris Bioscience.

Experimental design and statistical analyses

Electrophysiological data were collected using a Molecular Devices MultiClamp 700B amplifier with a HEKA instruments ITC-18 digitizer connected to a Dell computer running Windows 7, Axograph version 1.5.4, and LabChart (AD instruments). Statistical analyses were performed using Graphpad Prism. For behavioral experiments, 2-way repeated-measures ANOVAs with group (ethanol or water) as one independent between-subjects variable, and time as the second within-subjects independent variable were performed. If main effects but no interaction were found, groups were collapsed and analyzed either with unpaired t-tests or one-way ANOVAs with subsequent post hoc analysis (Tukey’s). One mouse with a history of ethanol drinking was excluded from analysis of cocaine-induced locomotor activity because it traveled 6.34, 8.88, and 5.08 standard deviations more during the first, second, and third 18 mg/kg cocaine sessions, respectively, than the mean distance traveled by the other cocaine-treated mice. Significance was set at P < 0.05.

Results

A history of ethanol drinking enhances methamphetamine-induced locomotion

To determine if a history of ethanol self-administration increases sensitivity to methamphetamine or cocaine, mice were given limited access to ethanol (n = 36) or water (n = 35) using the drinking in the dark procedure (Rhodes et al. 2005), followed by behavioral testing. Mice in the ethanol group consumed an average of 5.41 ± 0.08 g/kg/day with an average total intake of 75.8 ± 4.13 g/kg over the course of 14 days (Fig. 1B). Seven days following the last day of ethanol self-administration, mice were split into separate groups for assessment of saline- and either methamphetamine- or cocaine- induced locomotor activity (schematic, Fig. 1A,C,D). A preliminary session (S1) was conducted during which mice underwent a 15-minute habituation period in the locomotor chamber followed by saline injections every 15 minutes, resulting in three total injections during the hour session. There was no significant effect of a history of ethanol self-administration on locomotor activity during this saline session, in either the methamphetamine (n = 8 mice per group, F3,42 = 1.03, p = 0.39, data not shown) or cocaine (n = 7 mice per group, F3,36 = 0.27, p = .084, data not shown) groups. The first saline session was followed two, four, and six days later by methamphetamine or cocaine sessions during which locomotor activity was assessed using cumulative doses of methamphetamine (1, 3, and 5 mg/kg) or cocaine (3, 10, and 18 mg/kg).

Locomotor activity on the first day of methamphetamine administration (M1) was not different between mice who previously drank ethanol versus water (Fig. 2 A-C). Sensitization to methamphetamine was observed in both groups at 1 mg/kg methamphetamine, evidenced by increased locomotor stimulation in the second and third methamphetamine sessions compared to the first (Fig. 2A; 2-way repeated-measures ANOVA, main effect of session, F2,28 = 64.2, p < 0.0001, followed by one-way repeated-measures ANOVA, F2,30 = 63.2, p < 0.0001; Tukey’s M1 vs M2, p < 0.0001; M1 vs M3, p < 0.0001). At the 3 mg/kg methamphetamine dose there was no effect of ethanol drinking on locomotor stimulation or sensitization development over the 3 sessions (Fig. 2B; treatment-session interaction, F2,28 = 1.1, p = 0.35; main effect of session, F2,28 = 2.65, p = 0.088). This suggests that previous ethanol drinking does not alter acute sensitivity or the development of sensitization to low doses of methamphetamine.

Fig. 2. A history of ethanol drinking increases sensitivity to the locomotor-stimulating effects of methamphetamine but not cocaine.

Fig. 2.

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Locomotor activity elicited by 1 mg/kg methamphetamine increased from the first methamphetamine session (M1) to the second and third methamphetamine sessions (M2 and M3; A) in mice with a history of ethanol drinking (blue squares, EtOH) and water drinking controls (black circles, Water). Locomotor activity elicited by 3 mg/kg methamphetamine did not change across locomotor sessions and was not affected by a history of ethanol drinking (B). Locomotor activity elicited by 5 mg/kg methamphetamine was significantly increased in ethanol mice (C). Locomotor activity during the S2 (saline) session showing the average activity during the 15-minute habituation (labeled H) period and following the three saline (labeled S) injections was not affected by ethanol drinking in mice with repeated methamphetamine injections. (D, enlarged in inset). Locomotor activity elicited by 3 mg/kg cocaine increased from the first cocaine session (C1) to the second and third cocaine sessions (C2 and C3) and from C2 to C3 (E). Locomotor activity elicited by 10 mg/kg cocaine increased from C1 to C2 and C3, and from C2 to C3 (F). Locomotor activity elicited by 18 mg/kg increased from C1 to C2 and C3 (G). Locomotor activity during S2 was not differentially affected by ethanol drinking in mice with repeated cocaine injections (H, enlarged in inset). All data are presented as the change in locomotor activity during the 15-minute bin from the first saline treatment day (S1). Error bars represent ± standard error of the mean (SEM). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

While previous ethanol drinking did not alter the locomotor response to lower doses of methamphetamine, it did increase the locomotor response to a higher dose. Mice with a history of ethanol drinking exhibited a greater acute locomotor stimulation by 5 mg/kg methamphetamine compared to controls. Two-way repeated-measures ANOVA revealed no interaction (Fig. 2C; F2,28 = 0.69, p = 0.51) but did show a significant main effect of treatment (F1,14 = 6.79, p = 0.021), and an effect of treatment when days were collapsed (t23 = 4.3, p = 0.0002). The locomotor response to 5 mg/kg methamphetamine decreased in both groups (2-way ANOVA main effect of session, F2,28 = 31.7, p < 0.0001, followed by one-way repeated-measures ANOVA, F2,30 = 32.4, p < 0.0001; Tukey’s M1 vs M2, p < 0.0001; M1 vs M3, p < 0.0001), possibly due to methamphetamine-induced stereotypy (Nishikawa et al., 1983). These differences in locomotor activity did not appear to depend on conditioned behavior in response to i.p. injections or the locomotor chamber, as there was no significant difference between groups in response to i.p. injections of saline measured two days after the last methamphetamine session (Fig. 2D; S2, F3,42 = 1.0, p = 0.39). Together, these results suggest that a history of ethanol drinking enhances sensitivity to 5 mg/kg methamphetamine.

In contrast, a history of ethanol drinking did not impact the locomotor-stimulating effects of cocaine. There were no differences between ethanol- and water-drinking groups in cocaine-induced locomotor activity at any dose tested, (Fig. 2 E-G; 2-way repeated-measures ANOVAs, 3 mg/kg, interaction, F2, 24 = 1.83, p = 0.18, main effect of session, F2, 24 = 37.8, p < 0.0001; 10 mg/kg, interaction, F2, 24 = 0.44, p = 0.64, main effect of session, F2, 24 = 31.3, p < 0.0001; 18 mg/kg, interaction, F2, 24 = 0.079, p = 0.9243, main effect of session, F2, 24 = 11.8, p = 0.0003). Locomotor sensitization to cocaine proceeded similarly between groups, with activity increasing across sessions at 3 mg/kg (Fig. 2E; one-way ANOVA, F2, 26 = 35.5, p < 0.0001, Tukey’s C1 vs C2, p < 0.0001, C1 vs C3, p < 0.0001, C2 vs C3, p = 0.007), 10 mg/kg (Fig. 2F; F2, 26 = 32.7, p < 0.0001, Tukey’s C1 vs C2, p < 0.0001, C1 vs C3, p < 0.0001, C2 vs C3, p = 0.047), and 18 mg/kg cocaine (Fig. 2G; F2, 26 = 12.7, p = 0.0001, Tukey’s C1 vs C2, p = 0.0004, C1 vs C3, p < 0.0006). Furthermore, there was no difference between groups in response to i.p. injections of saline measured two days after the last methamphetamine session (Fig. 2H; 2-way repeated-measures ANOVA, F2,24 = 0.47, p = 0.63). While others have shown that repeated i.p injection of ethanol results in increased sensitivity to the locomotor-stimulating and rewarding effects of cocaine (Itzhak & Martin, 1999; Hopf et al., 2007; Bernier Whitaker, & Morikawa et al., 2011), here we report no cross-sensitization to cocaine following voluntary ethanol drinking.

A history of ethanol drinking blunts cellular effects of methamphetamine

Chronic ethanol exposure causes many adaptations in dopamine neuron physiology that may affect sensitivity to psychostimulants. While previous ethanol administration has been shown to either increase (Didone et al., 2016) or decrease dopamine neuron firing in vivo (Diana et al., 1996), there are multiple reports describing no change in basal dopamine neuron firing in brain slices ex vivo (Brodie, 2002; Hopf et al., 2007; Perra et al., 2011; Arora et al., 2013). However, this effect has only been investigated in the VTA. We therefore performed cell-attached recordings of SNc dopamine neurons and found that a history of ethanol drinking had no effect on basal firing rates (Fig. S2A; t93 = 0.13, p = 0.90) or reduction in firing by the dopamine D2 receptor agonist quinpirole in a sub-set of cells (Fig. S2B-D; 10 nM, t18 = 1.35, p = 0.19). We next investigated the effects of a history of ethanol drinking on DAT inhibitor-induced modulation of dendritic dopamine neurotransmission in SNc dopamine neurons. Dopamine neurons were voltage clamped at −55 mV, D2-IPSCs were measured (Beckstead et al. 2004), and methamphetamine applied by bath perfusion. Baseline D2-IPSC amplitudes were not significantly different between mice with a history of ethanol drinking and water drinking controls (average of 5 sweeps; water, 67.8 ± 11.9 pA, ethanol, 51.5 ± 5.46 pA, t16 = 1.25, p = 0.23, data not shown). As reported previously (Branch & Beckstead 2012), bath perfusion of a high concentration of methamphetamine (10 µM) caused an increase in the amplitude of D2-IPSCs (Fig. 3A,B,C). However, this effect was blunted in SNc dopamine neurons from mice that had previously drank ethanol (amplitude, average of 3 sweeps at peak effect of methamphetamine; control, n = 9 cells from 5 mice, 73.8 ± 7.6% increase; ethanol, n = 9 cells from 4 mice; 39.4 ± 5.8 %, t16 = 3.61, p = 0.002). Methamphetamine also produced an outward shift in the holding current (Branch & Beckstead, 2012), but this outward current was not affected by previous ethanol drinking (Fig. 4A,B,C; t10 = 0.57, p = 0.58;). Similarly, methamphetamine slowed the kinetics of D2-IPSCs, increasing half-width in a manner that is consistent with a decrease in DAT-mediated dopamine uptake. However, this effect was not altered by prior ethanol drinking (Fig. 4D,E; t12 = 1.37, p = 0.195). Interestingly, in the VTA, a previous history of ethanol drinking had no effect on methamphetamine-induced outward currents (t17 = 0.028, p = 0.98) or increases in the amplitudes (t19 = 1.6, p = 0.13) or widths (t19 = 0.39, p = 0.70) of D2-IPSCs (Fig. S3A-F). Together, these results suggest that while dopamine D2 receptor signaling and many cellular effects of methamphetamine remain intact, methamphetamine-induced enhancement of D2-IPSC amplitudes is selectively blunted in the SNc of mice with a history of ethanol drinking.

Fig. 3. A history of ethanol drinking blunts methamphetamine-induced enhancement of D2-IPSC amplitudes.

Fig. 3.

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Representative traces of D2-IPSC recordings from midbrain dopamine neurons before (black) and after (red) perfusion of methamphetamine (METH, 10 µM) in mice that previously were exposed to water (A1) or ethanol (A2). Time-course (B) and averaged (C) data from the peak effect (sweeps 8–10) of METH on D2-IPSC amplitudes showed significant blunting in mice with a history of ethanol drinking (blue squares, EtOH) compared to water-drinking controls (black circles, Water). Error bars represent ± SEM, **P < 0.01.

Fig. 4. A history of ethanol drinking has no effect on methamphetamine-induced increases in holding current or D2-IPSC widths.

Fig. 4.

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Traces of recordings from mice that consumed either water (black circles, Water) or ethanol (blue squares, EtOH) during perfusion of methamphetamine (METH, 10 µM). The dotted line depicts the baseline holding current (A). Time-course (B) and averaged (C) data from sweeps 8–10 of METH on the holding current showed no effect of ethanol drinking. Time-course (D) and averaged (E) data from sweeps 8–10 of METH on D2-IPSC widths (measured as the width of the IPSC at 50% of the peak amplitude) also showed no effect of ethanol drinking. Error bars represent ± SEM.

At high concentrations, methamphetamine acts as a substrate for the vesicular monoamine transporter VMAT2 (Sulzer et al. 1993) while low concentrations of methamphetamine increase the amplitude of D2-IPSCs by inhibiting DAT (Branch & Beckstead 2012). Thus, to determine if ethanol drinking alters VMAT2-independent effects of methamphetamine, we repeated the above experiments using a low concentration of methamphetamine (1 µM, Fig. 5A,B). Interestingly, ethanol drinking had no effect on low concentration methamphetamine-induced enhancement of D2-IPSCs (Fig. 5B; amplitude, average of 3 sweeps at peak effect of methamphetamine; control, n = 6 cells from 3 mice, 53.0 ± 5.4% increase; ethanol, n = 6 cells from 4 mice; 61.7 ± 9.9 %, t10 = 0.77, p = 0.46). We next tested the effect of a history of ethanol drinking on cocaine-induced enhancement of D2-IPSCs which, like low concentrations of methamphetamine, increases the width and amplitude of D2-IPSCs without engaging VMAT2 (Branch & Beckstead, 2012). Bath perfusion of a high concentration of cocaine caused an increase in D2-IPSC amplitudes (Fig. 5C,D) that was not blunted by ethanol history (Fig. 5D; change from baseline, average of 3 sweeps at peak effect; control, n = 11 cells from 5 mice; 118.6 ± 16.7 % increase; ethanol, n = 8 cells from 4 mice; 89.1 ± 11.1 %; t17 = 1.36, p = 0.19). Thus, the underlying neuroadaptations by which ethanol history blunts methamphetamine-induced increases in D2-IPSC amplitudes are consistent with VMAT2 as a locus of effect.

Fig. 5. A history of ethanol drinking has no effect on cocaine or low-concentration METH-induced enhancement of D2-IPSC amplitudes.

Fig. 5.

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Time-course (A) and averaged (B) data from the peak effect (sweeps 11–13) of METH (1 µM) on D2-IPSC amplitudes showed no effect of ethanol drinking. Time-course (C) and averaged (D) data from the peak effect (sweeps 10–12) of cocaine (10 µM) on D2-IPSC amplitudes also showed no effect of ethanol drinking (blue squares, EtOH) compared to water drinking controls (black circles, Water). Error bars represent ± SEM.

Discussion

Effects of prior ethanol drinking on methamphetamine-induced locomotion

Here we report that 14 days of ethanol consumption is sufficient to cause a long-lasting increase in the locomotor response to a high dose, but not low doses, of methamphetamine. This is in agreement with another recent study showing that a history of ethanol drinking does not increase methamphetamine-induced locomotor stimulation at doses smaller than 5 mg/kg (Fultz & Szumlinski 2018). Enhanced sensitivity to the locomotor stimulating effects of methamphetamine is consistent with studies showing that a history of ethanol drinking also enhances the rewarding effects of methamphetamine across a range of doses (0.5, 2, and 4 mg/kg) and increases consumption of liquid methamphetamine (Fultz et al., 2017; Fultz & Szumlinski, 2018). Here we also show that prior ethanol drinking does not alter the locomotor-stimulating effects of cocaine. This is in agreement with other studies showing that ethanol experience does not necessarily increase sensitivity to the locomotor-stimulating effects of cocaine (Lessov & Phillips, 2006; Manley & Little, 1997), at least not when tested two days following ethanol exposure. This is somewhat in contrast to research showing that repeated i.p. injections of ethanol in mice results in cross-sensitization to the locomotor-stimulating effects of cocaine (Itzhak & Martin, 1999) and in rats enhances conditioned place-preference for cocaine (Bernier, Whitaker, & Morikawa, 2011). However, this could be due to differences in the contingency/route of ethanol exposure (experimenter administered injection vs. self-administered consumption) and/or time from the last ethanol exposure (2 vs. 7 days). Overall, our results suggest that the time-course and route of ethanol administration used in this study captures a critical window of ethanol-induced plasticity that enhances sensitivity to methamphetamine but not cocaine.

Cellular effects of methamphetamine and cocaine

A history of ethanol drinking also increases sensitivity to the neuromodulatory effects of methamphetamine. While prior ethanol drinking enhances sensitivity to locomotor-stimulation by methamphetamine, here we show that methamphetamine-induced enhancement of dendritic dopamine neurotransmission in the SNc is depressed. This effect only occurs at a high concentration of methamphetamine, mirroring the increased locomotor-response to methamphetamine in mice with a history of ethanol drinking. Methamphetamine-induced modulation of dopamine neuron excitability and dopamine neurotransmission are complex. Low concentrations of methamphetamine (< 1 µM) activate an excitatory DAT-dependent ionic conductance and low to mid-range concentrations (0.1 – 3.0 µM) cause a prolonged increase in D2-IPSCs caused by a decrease in DAT-mediated clearance of dopamine (Branch & Beckstead 2012). At high concentrations (3 µM and above) methamphetamine can cause release of calcium from intracellular stores, alter potassium conductances, and decrease firing activity of dopamine neurons (Goodwin et al. 2009; Branch & Beckstead 2012; Lin, Sambo & Khoshbouei 2016). High concentrations of methamphetamine also decrease dopamine neurotransmission in part due to displacement of vesicular dopamine by acting as a substrate for VMAT2 (Sulzer et al. 1993; Goodwin et al. 2009; Yasumoto et al. 2009; Chu et al. 2010; Sulzer 2011; Lin et al. 2016). However, extracellular levels of dopamine remain high, likely due to efflux of dopamine through DAT (Goodwin et al. 2009). While methamphetamine has many cellular actions at high concentrations, the decreased methamphetamine-induced enhancement of D2-IPSC amplitudes reported here may be a result of increased action at VMAT2, with depletion of vesicular dopamine resulting in reduced dopamine release. This is consistent with research showing that ethanol drinking increases VMAT2 gene expression in the midbrain (Darlington et al. 2014). Unexpectedly, we observed no difference in the methamphetamine-induced outward current, which is also likely VMAT2 dependent (Sulzer 2011, Branch & Beckstead 2012). This could be due to the outward current being less sensitive than D2-IPSCs to methamphetamine action at VMAT2, or perhaps a ceiling effect of methamphetamine action at VMAT2, making reversal of DAT the determining step in eliciting outward currents under these experimental conditions. Indeed, the actions of amphetamine at VMAT2 and DAT are dissociable, as increasing cytosolic dopamine by inhibiting VMAT2 is not sufficient to drive dopamine efflux from terminals but does prime terminals for significantly shorter-latency of amphetamine-induced dopamine efflux due to action at DAT (Jones et al., 1998). Furthermore, there was no apparent change in the VMAT2-independent slowing of D2-IPSC kinetics by methamphetamine, which is instead mediated by the prolonged presence of dopamine due to decreased uptake by DAT (Branch & Beckstead 2012). This is in line with findings that DAT mRNA expression remains unchanged in both the SNc and VTA following ethanol drinking (Darlington et al., 2014). However, DAT expression and/or function is upregulated in other brain regions, most notably the striatum, after ethanol exposure (Itzhak & Martin, 1999; Budygin et al., 2006; Carrol et al., 2006; Healey, Winder, & Kash, 2008; Karkhanis et al., 2015). Interestingly, we observed no effect of a history of ethanol drinking on methamphetamine-induced enhancement of D2-IPSCs recorded from VTA neurons. We did observe subtle qualitative differences in the time-course and magnitude of methamphetamine induced-enhancement in the amplitude of D2-IPSCs in the SNc and VTA, even though the outward current and increase in D2-IPSC widths was similar between regions. This may be indicative of fundamental differences in the interaction of methamphetamine and VMAT2 in the context of dendritic dopamine neurotransmission in the SNc and VTA. The blunted action of methamphetamine-induced enhancement of D2-IPSC amplitudes in the SNc would result in decreased inhibition (disinhibition) of dopamine neurons (Aghajanian & Bunney 1977) thereby enhancing dopamine release in the striatum (Westerink, Kwint & deVries 1996; Kohl et al. 1998) and leading to an increased behavioral response to methamphetamine.

Interaction between ethanol, methamphetamine, and VMAT2

The behavioral effects of ethanol and methamphetamine may both depend in part on VMAT2 activity. The role of VMAT2 in the sensitivity to the reinforcing effects of ethanol have been explored in two studies of transgenic mice in which VMAT2 expression was knocked down. VMAT2 knockdown has been shown to either increase ethanol consumption (Hall, Sora & Uhl 2003) or decrease ethanol preference and consumption (Savelieva, Caudle & Miller 2006). Similar complex findings exist in the literature regarding VMAT2 contribution to the behavioral effects of methamphetamine. VMAT2 knockdown mice show enhanced sensitivity to low doses of methamphetamine (1 or 2 mg/kg) both during acute exposure and in the development of sensitization with repeated exposure (Fukushima et al. 2007). However, pharmacological VMAT2 inhibition decreases methamphetamine self-administration (Beckmann et al. 2012; Wilmouth et al. 2013). These discrepancies make it difficult to provide strong claims about the behavioral consequences of the blunted methamphetamine-induced enhancement of dopamine neurotransmission reported here. As repeated ethanol exposure increases VMAT2 gene expression (Darlington et al. 2014), and methamphetamine action at VMAT2 is thought to be responsible for blunting of D2-IPSCs (Branch & Beckstead 2012), it is possible that increased VMAT2 activity results in greater blunting of D2-IPSCs by methamphetamine. Thus, a history of ethanol drinking may increase both the cellular and behavioral effects of methamphetamine by increasing midbrain VMAT2 expression and decreasing D2-receptor mediated inhibition of dopamine neuron activity. Overall, the current findings contribute to our understanding of the complex interaction between ethanol and methamphetamine and may one day contribute to the development of treatment for co-occurrence of these substance use disorders.

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Acknowledgements

We would like to thank Mackenna Wollet and Will Lynch for assisting with the ethanol drinking procedure. This work was supported by National Institute of Health grants R01 DA32701 and AG052606 (to MJB) and a CoBRE PJI award (to ALS) as part of P20 GM125528, as well as funds from the Presbyterian Health Foundation and the Oklahoma Center for Adult Stem Cell Research, a program of TSET. The authors declare no competing financial interests.

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