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. Author manuscript; available in PMC: 2021 Aug 1.
Published in final edited form as: Eur J Neurosci. 2020 May 13;52(3):3110–3123. doi: 10.1111/ejn.14752

Astrocytic equilibrative nucleoside transporter type 1 upregulations in the dorsomedial and dorsolateral striatum distinctly coordinate goal-directed and habitual ethanol-seeking behaviors in mice

Sa-Ik Hong 1, Amanda Bullert 1,4, Matthew Baker 1, Doo-Sup Choi 1,2,3
PMCID: PMC7489292  NIHMSID: NIHMS1597492  PMID: 32306482

Abstract

Two distinct dorsal striatum regions, dorsomedial striatum (DMS) and dorsolateral striatum (DLS), are attributed to conditioned goal-directed and habitual reward-seeking behaviors, respectively. Previously, our study shows that the ethanol-sensitive adenosine transporter, equilibrative nucleoside transporter 1 (ENT1), regulates ethanol-drinking behaviors. Although ENT1 is expressed in both neurons and astrocytes, astrocytic ENT1 is thought to regulate adenosine levels in response to ethanol. However, the role of DMS and DLS astrocytic ENT1 in goal-directed and habitual ethanol-seeking is not well known. Here, we identified whether the upregulation of astrocytic ENT1 in the DMS and DLS differentially regulates operant seeking behaviors toward the 10% sucrose (10S), 10% ethanol and 10% sucrose (10E10S), and 10% ethanol (10E) in mice. Using 4 days of random interval (RI), mice exhibited habitual seeking for 10S, but goal-directed seeking toward 10E10S. Using the same mice conditioned with 10E10S, we examined 10E-seeking behavior on a fixed ratio (FR) for 6 days, RI for 8 days. On the other hand, during FR and the first 4 days of RI schedules, mice showed goal-directed seeking for 10E, whereas mice exhibited habitual seeking for 10E during the last 4 days of RI schedule. Interestingly, DMS astrocytic ENT1 upregulation promotes shift from habitual to goal-directed reward-seeking behaviors. By contrast, DLS astrocytic ENT1 upregulation showed no effects on behavioral shift. Taken together, our findings demonstrate that DMS astrocytic ENT1 contributes to reward-seeking behaviors.

Keywords: Equilibrative nucleoside transporter type 1, Dorsomedial striatum, Dorsolateral striatum, Goal-directed behavior, Ethanol

Graphical Abstract

graphic file with name nihms-1597492-f0008.jpg

1. Introduction

The ability to coordinate goal-directed and habitual behaviors is critical for the decision-making process, which requires fine-tuning and neurofeedback. Goal-directed controls assign value to action through the action-outcome contingency by finely evaluating the rewards in outcome devaluation. Habitual controls assign values of actions with stimulus-response contingency and are less engaged in the outcome-based evaluation (Rangel et al., 2008). Habitual behavior upon repeated conditioning is a hallmark of addictive disorders including alcohol use disorder (AUD) (Yin & Knowlton, 2006; Rangel et al., 2008). The dorsal striatum encodes flexibility of outcome devaluation after the establishment of motivational properties by the ventral striatum (nucleus accumbens)-associated limbic network (Everitt & Robbins, 2005; Yin & Knowlton, 2006; Koob & Volkow, 2010). More specifically, the dorsomedial striatum (DMS) governs sensitive and goal-directed evaluation, whereas the dorsolateral striatum (DLS) regulates resistant and habitual responses to outcome devaluation (Yin & Knowlton, 2006; Jahanshahi et al., 2015; Lovinger & Alvarez, 2017).

Adenosine is a fine neuromodulator in the brain through the synchronizing and desynchronizing of several neurotransmitter-dependent neuronal activities (Sebastiao & Ribeiro, 2000). Consequently, the dysregulation of adenosine signaling is implicated in psychiatric and neurological diseases (Burnstock, 2008; Cheffer et al., 2018). Equilibrative nucleoside transporter type 1 (ENT1), the bidirectional transporter of purines and pyrimidines including adenosine, is widely expressed throughout the brain including the DMS and DLS (Anderson et al., 1999; Pastor-Anglada & Perez-Torras, 2018). Interestingly, astrocytes regulate extracellular or synaptic adenosine levels partially through ENT1 (Nagai et al., 2005; Tanaka et al., 2011; Boddum et al., 2016; Cronstein & Sitkovsky, 2017; Cheffer et al., 2018). ENT1-mediated adenosine tone dysfunction is associated with various neuropsychiatric disorders such as anxiety, depression, maladaptive impulsivity, and AUD (Choi et al., 2004; Kaster et al., 2015; Serchov et al., 2015; Oliveros et al., 2017; Cheffer et al., 2018). AUD is of particular interest, as ENT1 is an ethanol-sensitive adenosine transporter and responsible for the regulation of ethanol-mediated adenosine levels. Interestingly, acute ethanol treatment increases adenosine levels by inhibiting ENT1, which contributes to the intoxicating effect of ethanol. However, genetic ablation of ENT1 decreases adenosine levels and increases ethanol preference, drinking and withdrawal seizures (Choi et al., 2004; Asatryan et al., 2011; Sharma et al., 2018; Jia et al., 2019), which mimics AUD patients who are tolerant to intoxicating effects of alcohol upon repeated alcohol exposure (Melendez & Kalivas, 2004). Yet, it has not been studied whether overexpression of astrocytic ENT1 in the DMS and DLS regulates operant ethanol-seeking behaviors.

In this study, we employed viral fluorescence-tagged ENT1 delivery to increase region- and astrocyte-specific expression of ENT1. We utilized three kinds of reward outcomes to identify how astrocytic ENT1 upregulation may contribute to reward type-dependent seeking behaviors: sucrose reward, ethanol-containing reward, and ethanol reward. In addition, we examined whether astrocytic ENT1 upregulations in the DMS and DLS distinctly regulate reward-seeking behaviors in the different operant schedules (ratio schedule for goal-directed behavior and interval schedule for habitual behavior). Our study elucidates the roles of DMS and DLS ENT1 up-regulation in the goal-directed and habitual reward-seeking behaviors.

2. Materials and methods

2.1. Animals

Male C57BL/6J mice were purchased from Jackson Laboratory (Bar Harbor, ME, USA). Mice were housed in standard Plexiglas cages. The colony room was maintained at a constant temperature (24 ± 1°C) and humidity (60% ± 2%) under a 12:12 h light/dark cycle with lights on at 0700 and lights off at 1900. Mice aged between 8–10 weeks old were used for all experiments. Mice were allowed ad libitum access to food chow and water. For the operant conditioning, mice were weight restricted to 85–90% of their baseline weight, at which they were maintained for the duration of experimental procedures. The total number of mice in all experiments was 176 (60 for the radioligand binding assay, 18 for immunohistochemistry, 20 for Western blot analysis, 40 for sucrose-seeking, 38 for ethanol-containing reward- and ethanol-seeking experiments). All experimental procedures were approved by the Mayo Clinic Institutional Animal Care and Use Committee and performed in accordance with NIH guidelines.

2.2. Virus

Viruses were purchased from Vector Biolabs (Malvern, PA, USA). The concept for the designed structure was similar to the virus used in our previous paper with a fluorescence proteinfused ENT1 that still successfully enables transporter’s activity (Jia et al., 2019). We injected viruses at the following titers: AAV5-glial fibrillary acidic protein (GFAP)-mCherry for the control group, 1.6 × 1013 GC/ml; AAV5-GFAP-ENT1/mCherry for the overexpression group, 8.2 × 1012 GC/ml.

2.3. Stereotaxic surgery

Mice were anesthetized with isoflurane (1.5% in oxygen gas) with the VetFlo™ vaporizer with a single-channel anesthesia stand (Kent Scientific Corporation, Torrington, CT, USA) and placed on the digital stereotaxic alignment system (Model 1900; David Kopf Instruments, Tujunga, CA, USA). Hair was trimmed and the skull was exposed using an 8-gauge electrosurgical skin cutter (KLS Martin, Jacksonville, FL, USA). The skull was leveled using a dual-tilt measurement tool. Holes were drilled in the skull at the appropriate stereotaxic coordinates. Viruses were infused at 100 nl/min (AP +0.5 mm, ML ±1.2 mm, DV −2.0 mm from the dura for DMS; AP +0.5 mm, ML ±2.2 mm, DV −2.0 mm from the dura for DLS) for 5 min through a 35-gauge injection needle (cat #NF35BV; World Precision Instruments, Sarasota, FL, USA) using a microsyringe pump (Model UMP3; World Precision Instruments). The injection needle remained in place for an additional 5 min following the end of the injection. Following stereotaxic surgery, we injected buprenorphine sustained-release LAB (1 mg/kg, s.c.; ZooPharm, Laramie, WY, USA) to alleviate post-surgery pain. Mice were utilized for the experiments 4–5 wk after the virus injection. We referred to the literature (Paxinos and Franklin, 2001) for the coordinates and modified brain maps.

2.4. Operant reward-seeking behavior test

As illustrated in Fig. 1, we employed the same operant chambers and modified operant schedules from our previous studies (Nam et al., 2013b; Hong et al., 2019a; Hong et al., 2019b). Briefly, mice were placed in two-hole operant chambers (Med-Associates, St Albans, VT, USA) where they poke a single active hole for a reward (10 μl per a reinforcer). Another hole (inactive) did not respond to the animal’s action. The rewards were dissolved in tap water. On the first day for magazine training, mice were conditioned to approach the magazine on a random time schedule with a reward delivered for 30 min regardless of nose-poking behaviors. Next, mice were conditioned on the fixed ratio 1 (FR1) schedule with either sucrose (10S) or ethanol-containing reward (10E10S; v/v for ethanol) for 1 h. Except for the FR1 schedule with 10E10S or 10S reward and the extinction test, all the operant schedules with the rewards were 30 min in duration. However, if mice received a total of 60 reward outcomes, operant sessions were immediately terminated regardless of the remained session time duration. In the FR1/2/3 schedules, one reward followed 1/2/3 nose-pokes. In random interval 30/60/120 (RI30/60/120) schedules, there was a time-out interval for average 30/60/120 sec between two active nose-pokes. Only after a rewarded nose-poke, the cue sound and light were exhibited for 2 sec. On the evaluation tests, mice were given 1 h of ad libitum access to food chow (for the valued state, V) or the reinforcer in the operant chamber (for the devalued state, DV) and then underwent the extinction test for 10 min in the same conditioning schedule with the previous schedule. The order of the valued and devalued states was counterbalanced across mice. Immediately after the extinction test, to reinstate the conditioned reward-seeking behaviors, we performed the same conditioning schedule with the previous schedule on the same day and mice received the reward. Latency to the magazine (sec) indicates latency time from nose-poking the hole to approaching the magazine.

Fig. 1.

Fig. 1

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Experimental scheme. Using two-hole nose poking operant chambers, we examined 10S, 10E10S, or 10E reward-seeking behaviors.

1). 10S or 10E10S reward-seeking behaviors.

Two-hole operant chamber delivered either 10S or 10E10S reward following a rewarded nose-poke. After the FR1 schedule with 4 sessions, mice were conditioned on the shortened RI schedule (RI30 with 1 session → RI 60 with 3 sessions). The next day, we performed the evaluation tests. We presented the food chow (for the valued state) or 10S (for the devalued state) in mice conditioned with the 10S reward, or we presented the food chow (for the valued state) or 10E10S (for the devalued state) in mice conditioned with the 10E10S reward.

2). 10E reward-seeking behaviors.

Two-hole operant chamber delivered 10E reward following a rewarded nose-poke. We employed the same cohort of mice after the evaluation test with 10E10S reward and conditioned them with 10E. Mice received time for consuming unlimited food chow in the home cage 1 h before the operant conditioning with 10E, so we developed the valued state for seeking ethanol only. At first, we conditioned mice on the FR schedule with the 10E reward: FR1 with 3 sessions → FR2 with 1 session → FR3 with 2 sessions. The next day, we performed the evaluation tests. We presented the food chow (the valued state) or 10E (the devalued state). One day after the evaluation tests, we conditioned mice on RI schedule with the 10E reward: shortened RI schedule (RI30 with 1 session → RI60 with 3 sessions) and then extended RI schedule (additional RI120 4 sessions). We performed the evaluation test (EV#1) after the shortened RI schedule and the evaluation test (EV#2) after the extended RI schedule. We presented the food chow (the valued state) or 10E (the devalued state).

2.5. Radio-ligand binding assay

To measure the ENT1 protein level, the ENT1 binding was measured using [3H]-nitrobenzylthioinosine (NBTI; Moravek Inc., Brea, CA, USA) binding assay as described previously (Choi et al., 2004; Kim et al., 2011; Jia et al., 2019). Brain tissue from five mice was pooled as one sample (n = 1) to obtain sufficient protein amounts for the binding assay in the DMS and DLS. To ensure the ENT1 expression, we performed the binding assay with five pooled samples (n = 6, brain tissues from 30 mice). Brain samples were homogenized in 10% sucrose solution with protease inhibitor and centrifuged at 1,000 g for 4 min. The supernatant was carefully collected and centrifuged again at 10,000 g for another 10 min, and then the pellet was resuspended in the 50 mM Tris-HCl buffer (Bio-Rad, Hercules, CA, USA). [3H]-NBTI saturation (20 nM) binding assays were performed at 37 °C in 10 mM Tris-HCl for 45 min with dilazep (ENT1 inhibitor, 10 μM; Tocris) for non-specific binding or without diazep for total binding. Binding reactions were terminated by filtration with ice-cold Tris-HCl buffer through Whatman GF/B paper using a cell harvester (Inotech Biosystems International Inc., Brandon, FL, USA). The membrane-bound radio-ligand was measured using scintillation spectrometry (Beckman Coulter Inc., Brea, CA, USA). Specific [3H]-NBTI binding was determined by substituting nonspecific binding from total binding values.

2.6. Image collection and analysis of astrocyte process

Brains were fixed with 4% paraformaldehyde (Sigma-Aldrich, St. Louis, MO, USA) and transferred to 30% sucrose (Sigma-Aldrich) in phosphate-buffered saline at 4°C for 72 h. Brains were then frozen in dry ice and sectioned at 40 μm using a microtome (Leica Corp., Bannockburn, IL). Brain slices were stored at −20°C in a cryoprotectant solution containing 30% sucrose (Sigma-Aldrich), 30% ethylene glycol (Sigma-Aldrich) in phosphate-buffered saline. Sections were incubated in 0.2% Triton X-100 (Sigma-Aldrich), 5% bovine serum albumin in phosphate-buffered saline for 1 h followed by incubation with the primary antibody in 5% bovine serum albumin overnight at 4°C. Primary antibodies that were used in the present study included mouse anti-GFAP Alexa Fluor 488 [1:100, monoclonal immunoglobulin G1 (IgG1), #53-9892-82; Thermo Fisher, Waltham, MA, USA] antibody. After three times of washing with phosphate-buffered saline, the sections were mounted onto a glass slide coated with gelatin and cover-slipped with a VECTASHIELD® antifade mounting medium with DAPI (4’,6-diamidino-2-phenylindole) (Vector Laboratories, Burlingame, CA, USA). Images were obtained using an LSM 700 laser scanning confocal microscope (Carl Zeiss, Heidelberg, Germany) using a 10x lens (Fig. 2b left 2 panels), a 100x oil-immersion lens (Fig. 2b right 6 panels), and 63x water-immersion lens (Fig. 3b–f) for Simple Neurite Tracer and Sholl analysis.

Fig. 2.

Fig. 2

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Effects of ENT1 delivery on protein expressions of ENT1 and adenosine receptors in the DMS and DLS. (a) [3H]-NBTI binding in the DMS and DLS of mice (n = 6/group). (b) Expressions of mCherry-tagging ENT1 (magenta), GFAP (green), and DAPI (blue) in the DMS (top) and DLS (bottom). Protein expressions of (c) A1R and (d) A2AR in the DMS and DLS of mice (n = 5/group). Data represented as mean ± SEM. *p < 0.05, comparing the control group of each brain region. Student’s unpaired t-test. Ctrl, control group; OX, overexpression group.

Fig. 3.

Fig. 3

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Effects of DMS and DLS astrocytic ENT1 upregulation on GFAP expression and morphology. (a) Changes in GFAP protein expressions in the DMS and DLS of mice (n = 5/group). (b) Representative images of 3D astrocytic morphology reconstruction with Simple Neurite Tracer. Changes in (c) the number of astrocyte process, (d) total length of astrocyte processes, (e) astrocyte process thickness, and (f) maximum extension of astrocyte process in the DMS and DLS of mice (DMS ctrl group, n = 15; DMS OX group, n = 16; DLS ctrl group, n = 15; DLS OX group, n = 20). Data represented as mean ± SEM. *p < 0.05, comparing the control group of each brain region. Student’s unpaired t-test. Ctrl, control group; OX, overexpression group.

GFAP+ cells were traced in 3D using the Simple Neurite Tracer plugin (FIJI, NIH Image/J) and the maximum extension (μm) of astrocyte process from the nucleus were measured using the Sholl analysis function of Simple Neurite Tracer, as described (Ferreira et al., 2014; Molumby et al., 2016; Tavares et al., 2017).

2.7. Western blot analysis

Tissue containing the DMS and DLS was punched out from coronal slices (500-μm thick) and homogenized in a Storm 24 Bullet Blender (Next Advance, Averill Park, NY, USA) for 3 min at a speed setting of 4, with 0.5 mm zirconium oxide beads in combination with Cell-lytic MT mammalian tissue extraction buffer (Sigma-Aldrich) containing a protease inhibitor cocktail (Sigma Aldrich). Equal amounts of protein extracts (20 μg) were denatured and subjected to SDS-polyacrylamide gel electrophoresis. Protein-separated PVDF membranes (Bio-Rad, Philadelphia, PA, USA) were blocked with 5% bovine serum albumin in TBST [(in mM) 24 Tris, pH 7.4, 137 NaCl, 2.7 KCl, and 0.05% Tween 20] for 1 h at room temperature. These membranes were then incubated with rabbit anti-A1R (adenosine A1 receptor) (1:500, polyclonal IgG, #AAR-006; Alomone Labs, Jerusalem, Israel), rabbit anti-A2AR (adenosine A2A receptor) (1:500, polyclonal IgG, #AAR-002; Alomone Labs, Jerusalem, Israel), mouse anti-GFAP (1:500, monoclonal IgG1, #3670; Cell Signaling Technology, Danvers, MA, USA), and mouse anti-GAPDH (glyceraldehyde 3-phosphate dehydrogenase) (1:2000, monoclonal IgG1 clone 6C5, #MAB374; Millipore, Burlington, MA, USA) antibodies overnight at 4 °C. After washing with TBST, the membranes were incubated for 1 h with Goat anti-rabbit IgG-HRP (1:2500, #7074, Cell Signaling Technology) and Horse anti-mouse IgG-HRP conjugated antibodies (1:2500 for anti-GFAP antibody, 1:5000 for anti-GAPDH antibody, #7076, Cell Signaling Technology) at room temperature. The proteins were visualized by ECL solution (Azure, Dublin, CA, USA) using the Azure 300 Chemiluminescent Western Blot Imaging System (Azure).

2.8. Statistical analysis

All data represented as the mean ± standard error of the mean (SEM) and were analyzed by unpaired/paired two-tailed Student’s t-tests, one-way analysis of variance (ANOVA)/repeated measures one-way ANOVA followed by Bonferroni’s multiple comparisons tests, and two-way ANOVA/repeated measures ANOVA followed by Bonferroni’s multiple comparisons tests using Prism 7.0 (GraphPad Software Inc., San Diego, CA, USA). Statistical significance was set at p < 0.05.

3. Results

3.1. Effects of viral ENT1 delivery on the expressions of ENT1 and adenosine receptors in the DMS and DLS

To validate ENT1 expression change, we isolated the brain tissue after 4 weeks of virus injection. We utilized AAV with the GFAP promoter-driven ENT1 (mCherry fluorescence protein fused to C-terminal of mouse ENT1) targeted to GFAP, an astrocyte marker, to identify the location of ENT1 expression. We employed the radio-ligand binding assay to examine whether this virus technique increases ENT1 protein. Viral ENT1 delivery successfully enhanced specific [3H]-NBTI binding in the DMS (Fig. 2a left; t = 0.917, p < 0.001, n = 6/group) and DLS (Fig. 2a right; t = 4.498, p = 0.001, n = 6/group). As expected, mCherry was expressed in GFAP-expressing cells in the DMS (Fig. 2b top) and DLS (Fig. 2b bottom). Thus, these results imply that our viral delivery increased astrocytic ENT1 in the DMS and DLS of mice.

Furthermore, to identify the protein expression changes of adenosine receptors (A1R and A2AR) we employed Western blot analysis 4-week after the viral astrocytic ENT1 delivery. Astrocytic ENT1 overexpression did not alter the protein expression levels of A1R in the DMS (Fig. 2c left; t = 0.479, p = 0.645, n = 5/group) and DLS (Fig. 2c right; t = 1.026, p = 0.335, n = 5/group). However, astrocytic ENT1 upregulation increased the protein expression levels of DMS A2AR (Fig. 2c left; t = 2.914, p = 0.020, n = 5/group), but it reduced those of DLS A2AR (Fig. 2c right; t = 4.385, p = 0.002, n = 5/group). These results suggest that astrocytic ENT1 regulates A2AR expression rather than A1R in the DMS and DLS.

3.2. Effects of DMS and DLS astrocytic ENT1 upregulation on GFAP protein expression and astrocyte morphology

Next, we investigated whether astrocytic ENT1 delivery itself regulates astrocyte density and morphology since ENT1 deletion reduces striatal GFAP expression level (Hinton et al., 2014). Astrocytic ENT1 upregulation increased the GFAP protein density in the DMS (Fig. 3a left; t = 4.767, p = 0.001, n = 5/group) and DLS (Fig. 3a right; t = 3.235, p = 0.012, n = 5/group). Then, to further examine astrocytic ENT1-driven changes in astrocyte morphology, we traced the imaging with the fluorescence dye-conjugated GFAP antibody from four treatment groups: DMS control group (n = 15), overexpression group (n = 16); DLS control group (n = 15), overexpression group (n = 20). Astrocytic ENT1 overexpression increased the number of astrocyte process in the DMS (Fig. 3c left; t = 2.341, p = 0.026) and DLS (Fig. 3c right; t = 2.637, p = 0.013). However, astrocytic ENT1 overexpression did not change total length of process (for DMS, Fig. 3d left, t = 1.560, p = 0.130; for DLS, Fig. 3d right, t = 1.736, p = 0.092), process thickness (for DMS, Fig. 3e left, t = 0.569, p = 0.574; for DLS, Fig. 3e right, t = 0.403, p = 0.690), and maximum extension of process (for DMS, Fig. 3f left, t = 0.467, p = 0.644; for DLS, Fig. 3f right, t = 1.215, p = 0.233). These results suggest that DMS and DLS ENT1 overexpression increases GFAP protein expression and alters astrocyte morphology.

3.3. Effects of DMS and DLS astrocytic ENT1 upregulation on sucrose reward-seeking behaviors

For the behavioral experiments, we employed the operant conditioning with the 10S reward in the shortened RI schedule to identify the difference of reward evaluation between the sucrose and ethanol-containing rewards and the effects of astrocytic ENT1 upregulation on sucrose reward-seeking behaviors. Astrocytic ENT1 upregulation in the DMS increased nose-poking behaviors in the acquisition sessions [Fig. 4a, F(1,18) = 4.559, p = 0.047, n = 10/group] and the valued state of the extinction test (Fig. 4b, t = 2.889, p = 0.010, n = 10/group), but not in the DLS [Fig. 4a, F(1,18) = 0.104, p = 0.751, n = 10/group; Fig. 4b, t = 0.959, p = 0.350, n = 10/group]. Furthermore, DMS astrocytic ENT1 upregulation produced nose-poking changes between the valued and devalued states (Fig. 4c left, t = 2.711, p = 0.024, n = 10/group), while mice showed no difference of nose-poking behaviors (Fig. 4c left, t = 1.054, p = 0.319, n = 10/group). DLS astrocytic ENT1 upregulation, however, maintained reward evaluation (Fig. 4c right; control group, t = 0.376, p = 0.716, n = 10/group; overexpression group, t = 1.180, p = 0.268, n = 10/group).

Fig. 4.

Fig. 4

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Effects of DMS and DLS astrocytic ENT1 upregulation on 10S-seeking behaviors. (a) Nose-poking behaviors toward the 10S rewards in the RI schedule of the operant conditioning. (b) Nose-poking behaviors toward the 10S rewards in the valued states of the extinction test. (c) Changes in nose-poking behaviors toward the 10S rewards between the valued (V) and devalued (DV) states. n = 9/group for DMS groups in 10E10S-seeking, n = 10/group for DLS groups in 10E10S-seeking, n = 10/group for DMS and DLS groups in 10S-seeking. Data represented as mean ± SEM. *p < 0.05, comparing (a, b) control (Ctrl) and overexpression (OX) groups, (c) the valued and devalued states. (a) Two-way repeated measures ANOVA followed by Bonferroni’s multiple comparisons test, (b) Student’s unpaired t-test, (c) Student’s paired t-test.

3.4. Effects of DMS and DLS astrocytic ENT1 upregulation on ethanol-containing reward-seeking behaviors

Previously, we showed that the shortened RI schedule in an operant chamber increase voluntarily reward-seeking for 10E10S compared to 10S in the Y-maze (Hong et al., 2019a; Hong et al., 2019b). Thus, we examined whether the shortened RI schedule with the 10E10S reward develops goal-directed or habitual seeking behaviors and how astrocytic ENT1 upregulation contributes to this reward evaluation. Four weeks after the virus injection, we conditioned mice with the 10E10S reward in the shortened RI schedule. In the operant conditioning sessions [Fig. 5a; DMS, F(1,16) = 0.340, p = 0.568, n = 9/group; DLS, F(1,18) = 0.507, p = 0.486, n = 10/group] and the valued state of the extinction test (Fig. 5b; DMS, t = 0.298, p = 0.770, n = 9/group; DLS, t = 0.684, p = 0.503, n = 10/group), DMS and DLS ENT1 upregulation did not alter nose-poking behaviors. Interestingly, mice showed different nose-poking behaviors between the valued and devalued states in the evaluation test (Fig. 5c; DMS, t = 8.382, p < 0.001, n = 9/group; DLS, t = 3.950, p = 0.003, n = 10/group) and astrocytic ENT1 upregulation in either DMS or DLS maintained the reward evaluation (Fig. 5c; DMS, t = 4.472, p = 0.002, n = 9/group; DLS, t = 4.851, p = 0.001, n = 10/group). These results suggest that the operant conditioning with 10E10S in the shortened RI schedule promotes goal-directed reward-seeking behaviors regardless of DMS and DLS astrocytic ENT1 upregulation.

Fig. 5.

Fig. 5

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Effects of DMS and DLS astrocytic ENT1 upregulation on 10E10S-seeking behaviors. (a) Nose-poking behaviors toward the 10E10S rewards in the RI schedule of the operant conditioning. (b) Nose-poking behaviors toward the 10E10S rewards in the valued states of the extinction test. (c) Changes in nose-poking behaviors toward the 10E10S rewards between the valued (V) and devalued (DV) states. n = 9/group for DMS groups in 10E10S-seeking, n = 10/group for DLS groups in 10E10S-seeking, n = 10/group for DMS and DLS groups in 10S-seeking. Data represented as mean ± SEM. *p < 0.05, comparing (a, b) control (Ctrl) and overexpression (OX) groups, (c) the valued and devalued states. (a) Two-way repeated measures ANOVA followed by Bonferroni’s multiple comparisons test, (b) Student’s unpaired t-test, (c) Student’s paired t-test.

Therefore, these findings imply that the operant conditioning with the 10S reward in the shortened RI schedule may develop habitual reward evaluation unlike conditioning with 10E10S as the reward. Additionally, the upregulation of astrocytic ENT1 in the DMS but not the DLS may heighten the motivation for sucrose-seeking while promoting goal-directed seeking.

3.5. Effects of DMS and DLS astrocytic ENT1 upregulation on operant ratio schedule with ethanol

Transition from the 10E10S to 10E rewards without a sucrose fading method successfully establishes the value of ethanol as the reward (Hong et al., 2019b). Thus, we conditioned mice with the 10E reward to investigate the role of DMS and DLS astrocytic ENT1 in ethanol-seeking behaviors after the establishment of ethanol-containing reward-seeking behaviors. Although the FR schedules increased nose-poking behaviors [Fig. 6a; DMS, F(5,80) = 5.25, p = 0.001, n = 9/group; DLS, F(5,90) = 19.4, p < 0.001, n = 10/group], mice similarly sought the 10E reward during the FR schedule between the control and ENT1-upregulated groups [Fig. 6a; DMS, F(1,16) = 0.041, p = 0.841, n = 9/group; DLS, F(1,18) = 0.016, p = 0.900, n = 10/group]. In the valued state of the extinction test, DMS ENT1 upregulation did not change nose-poking behaviors (Fig. 6b left, t = 0.482, p = 0.823, n = 9/group), whereas DLS ENT1 upregulation decreased nose-poking behaviors (Fig. 6b right, t = 2.408, p = 0.027, n = 10/group). Additionally, ENT1 upregulation in the DMS (Fig. 6c; DMS control group, t = 2.917, p = 0.019, n = 9/group; DMS ENT1 overexpression group, t = 2.501, p = 0.037, n = 9/group) and DLS (Fig. 6c; DLS control group, t = 7.491, p < 0.001, n = 10/group; DLS ENT1 overexpression group, t = 7.915, p < 0.001, n = 10/group) did not change goal-directed reward evaluation. These results suggest that DLS ENT1 overexpression reduced the motivation for ethanol-seeking in mice. In addition, ENT1 upregulation in both DMS and DLS may maintain goal-directed ethanol-seeking behaviors.

Fig. 6.

Fig. 6

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Effects of DMS and DLS astrocytic ENT1 upregulation on the operant ratio schedule with 10E. (a) Nose-poking behaviors toward the 10E reward in the FR schedules of the operant conditioning. (b) Nose-poking behaviors toward the ethanol rewards in the valued states of the extinction test after the FR schedules. (c) Changes in nose-poking behaviors toward the 10E reward in the valued (V) and devalued (DV) states of the extinction test after the FR schedules. n = 9/group for DMS groups, n = 10/group for DLS groups. Data represented as mean ± SEM. *p < 0.05, comparing the valued and devalued states. (a) Two-way repeated measures ANOVA followed by Bonferroni’s multiple comparisons test, (b) Student’s unpaired t-test, (c) Student’s paired t-test. Ctrl, control group; OX, overexpression group.

3.6. Effects of DLS and DLS astrocytic ENT1 upregulation on operant interval schedule with ethanol

Following the FR schedule and the evaluation tests, we conditioned mice in the RI schedule to develop the habitual reward-seeking behaviors (Yin & Knowlton, 2006). We employed both shortened and extended RI schedules, and then performed the evaluation tests after each RI schedule to examine the transition from goal-directed to habitual ethanol-seeking behaviors. DMS astrocytic ENT1 upregulation mice showed similar seeking behaviors during the RI schedule [Fig. 7a, F(1,16) = 2.381, p = 0.142, n = 9/group] and in the valued states of the extinction tests (Fig. 7b; the evaluation test #1, t = 0.621, p = 0.543, n = 9/group; the evaluation test #2, t = 1.131, p = 0.275, n = 9/group). After the shortened RI schedule, DMS astrocytic ENT1 upregulation maintained goal-directed nose-poking behavior (Fig. 7c left side of the evaluation test #1, the control group, t = 7.129, p < 0.001, n = 9/group; Fig. 7c right side of the evaluation test #1, the overexpression group, t = 4.257, p = 0.003, n = 9/group). However, while the control group showed habitual reward evaluation after the extended RI schedule (Fig. 7c left side of the evaluation test #2, t = 1.573, p = 0.154, n = 9/group), DMS astrocytic ENT1 upregulation resulted in goal-directed reward evaluation (Fig. 7c right side of the evaluation test #2, t = 3.729, p = 0.006, n = 9/group). These results indicate that the shortened RI schedule may establish goal-directed ethanol-seeking, whereas the extended RI schedule may develop habitual ethanol-seeking behaviors with lower motivation. Furthermore, DMS astrocytic ENT1 upregulation may suppress the expression of habitual ethanol-seeking behaviors.

Fig. 7.

Fig. 7

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Effects of DMS and DLS astrocytic ENT1 upregulation on the operant interval schedule with 10E. Nose-poking behaviors toward the 10E reward in the (a for DMS groups, d for DLS groups) RI schedules of the operant conditioning. Nose-poking behaviors toward the ethanol rewards in the valued states of the extinction test after (b for DMS groups, e for DLS groups) RI schedules. Changes in nose-poking behaviors toward the 10E reward in the valued (V) and devalued (DV) states of the extinction test after (c for DMS groups, e for DLS groups) RI schedules. n = 9/group for DMS groups, n = 10/group for DLS groups. Data represented as mean ± SEM. *p < 0.05, comparing the valued and devalued states. (a, d) Two-way repeated measures ANOVA followed by Bonferroni’s multiple comparisons test, (b, e) Student’s unpaired t-test, (c, f) Student’s paired t-test. Ctrl, control group; OX, overexpression group.

Similar to the effects of DMS astrocytic ENT1 increase, DLS astrocytic ENT1 upregulation also caused similar seeking behaviors during the RI schedule [Fig. 7d, F(1,18) = 0.070, p = 0.793, n = 10/group] and in the valued states of the extinction tests (Fig. 7e; the evaluation test #1, t = 0.173, p = 0.865, n = 10/group; the evaluation test #2, t = 0.972, p = 0.344, n = 10/group) compared to the control virus group. After the shortened RI schedule, DLS astrocytic ENT1 upregulation did not change the goal-directed reward evaluation (Fig. 7f left side of the evaluation test #1, the control group, t = 2.759, p = 0.022, n = 10/group; Fig. 7f right side of the evaluation test #1, the overexpression group, t = 4.332, p = 0.002, n = 10/group). Even after the extended RI schedule, DLS astrocytic ENT1 upregulation did not change the habitual reward evaluation (Fig. 7f left side of the evaluation test #2, the control group, t = 2.058, p = 0.070, n = 10/group; Fig. 7f right side of the evaluation test #2, the overexpression group, t = 1.741, p = 0.116, n = 10/group). Thus, these results imply that DLS astrocytic ENT1 may not alter reward evaluation.

4. Discussion

The present study shows that DMS and DLS astrocytic ENT1 distinctly regulated the operant seeking behaviors toward the sucrose, ethanol, and sucrose-containing ethanol rewards. The addition of sucrose to ethanol facilitated goal-directed seeking behaviors after the same operant schedule. After the transition to a 10E reward, the FR schedule and the shortened RI schedule both exhibited goal-directed reward-seeking, whereas during the last four extended days of the RI schedule, mice displayed habitual seeking behaviors. DMS but not DLS astrocytic ENT1 upregulation promoted goal-directed sucrose-seeking with enhanced motivation. Meanwhile, DLS astrocytic ENT1 upregulation did not affect either goal-directed or habitual reward-seeking behaviors. Therefore, the astrocytic ENT1 in the DMS and DLS may differentially coordinate reward-seeking behaviors.

The ethanol-containing reward formed goal-directed behaviors unlike the behavioral effects of the sucrose reward in spite of the operant conditioning with the same operant schedule in this study. Consistently, mixing sucrose and ethanol can disturb habitual control and then yield goal-directed behavior in humans (Obst et al., 2018). These findings imply that ethanol exposure rather than a sucrose only reward may be associated with a distinguishable brain process for reward-seeking behaviors. Actually, additional evidence reveals that some drugs of abuse hijack normal reward processes and yield new brain processes to cause compulsive drug-seeking behaviors despite sharing the same reward pathway in the brain (Hyman & Malenka, 2001; Kelley & Berridge, 2002). Furthermore, striatal neurons separately respond to drugs versus sucrose rewards during goal-directed seeking behaviors and drug abstinence (Carelli, 2004; Cameron & Carelli, 2012), although encoding drug-seeking and natural reward-seeking can be commonly occurred in accordance with the brain regions and behavioral conditions (Pitchers et al., 2013; Pfarr et al., 2018). Thus, the brain networks associated with goal-directed and habitual behaviors may differentially encode the development of reward-seeking behaviors in terms of sucrose or ethanol-containing rewards. Furthermore, this reward type-dependent encoding may differently regulate manipulation-induced behavioral changes. For example, astrocytic ENT1 overexpression similarly changed behavioral flexibility toward the sucrose and ethanol rewards, but not the acquisition. This implies that manipulation-driven behavioral outcomes could be different according to the reward type, although ENT1 regulates two distinct behavioral processes, such as reward evaluation and acquisition of seeking behavior (Nam et al., 2013b).

The operant self-administration paradigm is widely used in preclinical addiction research since the paradigm utilizes voluntary drug-intake and requires cognitive processing and value-dependent decision-making (Sanchis-Segura & Spanagel, 2006). Thus, the operant ethanol self-administration paradigm directs either malleable or maintained ethanol-seeking behaviors according to the change in ethanol value. Interestingly, astrocytic ENT1 upregulation suppressed habitual ethanol evaluations but not goal-directed evaluations in our study. This infers that therapeutic approaches may differently affect ethanol-seeking depending on goal-directed or habitual ethanol-seeking since goal-directed and habitual seeking behaviors distinctly cause changes of brain structures and striatal neuronal activities (O’Hare et al., 2016; Lim et al., 2019). Accordingly, it may be beneficial to dissect drug-seeking behaviors into goal-directed or habitual behaviors when applying pharmacological and behavioral treatments (Hay et al., 2013; Stock, 2017).

Notably, our results indicate that astrocytic ENT1 overexpression in the DMS suppressed transition from goal-directed to habitual reward-seeking behaviors. Then, how are these motivated behaviors shifted between goal-directed and habitual seeking by repeated conditionings? A recent model suggests that repeated conditioning gradually shift toward DLS-mediated habitual control rather than DMS-mediated goal-directed control, and then exhibits habitual seeking behaviors (Bergstrom et al., 2018). Depending on the type of cellular manipulation such as genetically adapted modulation or changes only at a specific time point, it could be interpreted meaning that the manipulation leads to promote a shift from habitual to goal-directed actions or suppress a shift from goal-directed to habitual actions. Thus, in our study, astrocytic overexpression of ENT1 in the DMS may strengthen the goal-directed control, which may suppress a transition from goal-directed to habitual reward-seeking behaviors even with prolonged exposure to conditioned reward.

Meanwhile, it is quite interesting that DLS ENT1 upregulation did not alter both goal-directed and habitual seeking behaviors unlike the role of DMS ENT1. This could mean reward evaluation is regulated differently by ENT1-mediated adenosine signaling in the DMS and DLS.

Dorsal striatal astrocytes are structurally close to glutamatergic synapses rather than dopaminergic synapses (Octeau et al., 2018). Thus, astrocyte-mediated adenosine dynamics may intervene more in glutamatergic innervation, and extra ENT1 in the astrocytes may enable adenosine dynamics of the dopaminergic synapse as well as those of glutamatergic synapse more fluently. Following alterations in dopaminergic and glutamatergic innervations, the activities of direct and indirect pathways in the striatum may be distinctly regulated. It will be interesting to investigate the spatiotemporal ENT1-driven adenosine dynamics using neuronal imaging techniques with fluorescence-based adenosine sensors in various operant behavioral contexts such as between goal-directed and habitual behaviors.

Since ENT1 regulates both intracellular and extracellular adenosine levels bi-directionally, ENT1-mediated adenosine dynamics could change the expressions of adenosine-related receptors and transporters as non-specific ENT1 deletion reduces the expression of A2AR (Oliveros et al., 2017). Similarly, we found that astrocytic ENT1 drove DMS A2AR upregulation, DLS A2AR downregulation, and no changes in A1R of the DMS and DLS. Striatal astrocytic ENT1 may not intervene in A1R expression despite involvements between A1R and ENT1 (Jennings et al., 2001; Pinto-Duarte et al., 2005). In addition, this finding supports our previous research that ENT1 predominantly involves A2AR-dependent signaling (Nam et al., 2013a; Nam et al., 2013b). On the other hand, distinct regulatory direction for A2AR expression change between DMS and DLS may be shown because astrocytic ENT1-mediated adenosine tone may affect A2AR expression differently and adaptively according to spatial distribution changes of A2AR in the dorsal striatum (He et al., 2016). Furthermore, these adenosine system-related molecules could be differently tuned by synaptic ENT1 since astrocytic and synaptic ENT1 could have distinct roles depending on adenosine gradients of microcircuit environment (O’Donovan et al., 2018). Additionally, since synaptic ENT1 activity can be changed depending on the neuronal activity (Pinto-Duarte et al., 2005), different dynamics of cellular activity between neurons and astrocytes in even the same brain region may distinctly affect the ENT1-driven molecular changes. Thus, the study of the relationship between astrocyte- and synapse-specific ENT1 may be quite an interesting future topic.

Both A1R and A2AR contribute to operant seeking behaviors. Despite the lack of evidence for the effects of A1R activation on ethanol-seeking, A1R activation dampens operant seeking behaviors toward cocaine, methamphetamine (Hobson et al., 2013; Larson et al., 2019), while pharmacological and genetic blockade of A1R have no effect on ethanol self-administration and sucrose reward-seeking behaviors (Arolfo et al., 2004; Li et al., 2018). However, ENT1-mediated changes in seeking behaviors can be expected to be more A2AR-dependent rather than A1R since striatal ENT1-mediated A2AR signaling change may play an essential role in operant seeking behaviors (Nam et al., 2013a; Nam et al., 2013b) as discussed earlier. Interestingly, systemic A2AR inhibition promotes goal-directed sucrose-seeking (Li et al., 2018). Furthermore, genetic and pharmacological DLS A2AR inhibitions maintain sucrose-seeking behaviors (Nam et al., 2013b; Li et al., 2016). Consistently, our recent study shows that optogenetic inhibition of A2ARexpressing neurons in the DMS and systemic A2AR inhibition increase the value of ethanol, when A2AR agonist suppresses ethanol-seeking (Hong et al., 2019a). Meanwhile, optogenetic DLS A2AR activation and genetic DMS A2AR blockade induce goal-directed sucrose-seeking behaviors (Li et al., 2016). In addition, DMS A2AR activation suppresses goal-directed sucrose-seeking (Li et al., 2016) and the value of ethanol (Hong et al., 2019a; Hong et al., 2019b). Thus, DMS astrocytic ENT1-mediated behavioral changes in our study may be induced through DMS A2AR signaling.

Our findings uncover differences in behavioral responding to outcome evaluation toward the sucrose and ethanol rewards. Additionally, our results demonstrate that the repeated RI schedule develops habitual ethanol-seeking behaviors from goal-directed seeking. Moreover, we found the roles of DMS and DLS astrocytic ENT1 in ethanol-seeking. Overall, we provide a potential therapeutic strategy for suppressing habitual ethanol-seeking behaviors.

Highlights.

Using mice overexpressing adenosine transporter (ENT1) in astrocytes, we found that increased astrocytic ENT1 expression in the dorsomedial striatum (DMS) facilitated goal-directed reward-seeking behavior when mice were conditioned with either 10% sucrose or 10% ethanol. However, no changes were observed when ENT1 was over-expressed in astrocytes of the dorsolateral striatum (DLS). Our finding demonstrates that DMS astrocytic ENT1 contributes to reward-seeking behaviors.

ACKNOWLEDGEMENTS

This work was supported by the Samuel C. Johnson for Genomics of Addiction Program at Mayo Clinic, the Ulm Foundation, and National Institute on Alcohol Abuse and Alcoholism (AA018779).

Abbreviations

10E

10% ethanol

10E10S

10% ethanol and 10% sucrose

10S

10% sucrose

A1R

adenosine A1 receptor

A2AR

adenosine A2A receptor

ANOVA

one-way analysis of variance

AUD

alcohol use disorder

Ctrl

control group

DAPI

4’,6-diamidino-2-phenylindole

DLS

dorsolateral striatum

DMS

dorsomedial striatum

ENT1

equilibrative nucleoside transporter type 1

FR

fixed ratio

GAPDH

glyceraldehyde 3-phosphate dehydrogenase

GFAP

glial fibrillary acidic protein

IgG

Immunoglobulin G

NBTI

nitrobenzylthioinosine

OX

overexpression

RI

random interval

SEM

standard error of the mean

Footnotes

DECLARATIONS OF INTEREST

Dr. DS Choi is a scientific advisory board member to Peptron Inc., which had no role in preparation, review, or approval of the manuscript; nor decision to submit the manuscript for publication. All the other authors declare no biomedical financial interests or potential conflicts of interest. All other authors declare no conflict of interests.

DATA AVAILABILITY STATEMENT

Supporting data are available on request.

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