All experiments were performed in accordance with international guidelines for the care and use of experimental animals. ADAR2^flox/flox mice were maintained under a 12-h light-dark cycle (lights on from 08:00 to 20:00 h) with free access to water and food (Hideyama et al., 2010). Behavioral testing was performed between 9:00 and 18:00 h. After testing, the apparatus was cleaned with 70% ethanol to prevent bias caused by olfactory cues. All animal experiments, including production, maintenance protocols and behavioral studies, were reviewed and approved by the Animal Care and Use Committee of the Kyoto Prefectural University of Medicine (M26-250). Mice were anesthetized with a midazolam/medetomidine/butorphanol cocktail (4,0.3 and 5 mg/kg, respectively) during surgery. Prior to brain removal, all mice were euthanized with an excess of pentobarbital (50 mg/kg).

Male ADAR2^flox/flox mice with a C57BL/6J genetic background (10–13 weeks old) were anesthetized and mounted in a stereotaxic apparatus (Narishige, Tokyo, Japan). AAV-GFP/Cre (Addgene plasmid # 49056) was a gift from Dr. Fred Gage (Kaspar et al., 2002). pAAV-GFP control vector was purchased from Cell Biolabs (San Diego, CA, USA). For recombinant AAV (rAAV) production, the vectors were cotransfected into HEK293 cells with pAAV-DJ and pHelper (Cell Biolabs), according to the manufacturer’s instructions. rAAV particles were purified by discontinuous step gradient using iodixanol (Opti-Prep; Nycomed Pharma, Oslo, Norway), as described previously (Aoki et al., 2016). Iodixanol fractions were concentrated and dialyzed by centrifugation through a Biomax 100K filter (Millipore, Billerica, MA, USA) with phosphate-buffered saline (PBS) containing 5% sorbitol. rAAV titers were determined by quantitative PCR and aliquoted rAAVs were stored at −80°C. AAV-GFP/Cre (1.63 × 10¹³ viral genome particles/ml) or AAV-GFP (2.2 × 10¹² viral genome particles/mL) containing Fast Green dye (Nacalai Tesque, Kyoto, Japan) was bilaterally microinjected (1 μl/site) into the NAc (AP, +2 mm from Bregma; ML, ±0.8 mm from midline; DV, +4.3 mm below the skull surface) using a 30-gauge Hamilton syringe needle (Hamilton, Reno, NV, USA) at a rate of 0.2 μl/min. The needle was left in place for 12 min after each injection before slow retraction. GFP/Cre or GFP expression was allowed to develop for 4 weeks. After behavioral analysis, GFP/Cre and GFP gene expression were confirmed by immunohistochemical analysis using anti-GFP antibodies (MBL, Nagoya, Japan). Genomic DNA from the NAc was prepared using the NucleoSpin TriPrep kit (MACHEREY-NAGEL, Düren, Germany). Primers (5′-CTGGTTCATAACAGATCCTCAGGG-3′ and 5′-GTCTCCCTTGTCCTTCCAGGTAGC-3′) were used for genomic ADAR2 PCR (Hideyama et al., 2010).

Under deep anesthesia, AAV-GFP/Cre- or AAV-GFP- injected mice (n = 5) were perfused via the left cardiac ventricle with 20 ml chilled 0.1 M PBS followed by 40 ml of fixative containing 4% paraformaldehyde in 0.1 M phosphate buffer. Brains were post-fixed in the same fixative at 4°C overnight. After cryoprotection in 25% sucrose in 0.1 M PB for 48 h at 4°C, coronal sections (10-μm thick) were cut using a cryostat and mounted on coated glass slides (Fisherbrand Superfrost Plus; Fisher Scientific, Hampton, NH, USA). Sections were exposed to an antigen retrieval solution (Histo VT One; Nacalai Tesque, Kyoto, Japan) for 20 min at 70°C. After washing in PBS and incubating in blocking solution (Blocking One Histo; Nacalai Tesque, Kyoto, Japan), sections were incubated with mouse monoclonal anti-ADAR2 antibody (1:200; sc-73409, Santa Cruz Biotechnology Inc., Dallas, TX, USA) and rabbit monoclonal anti-GFP antibody (1:2,000; MBL, Nagoya, Japan) for 24 h at 4°C. For detection of primary antibodies, Alexa 594-conjugated goat anti-mouse and Alexa 488-conjugated goat anti-rabbit antibodies (1:500; Life Technologies, Carlsbad, CA, USA) were used. For NeuN detection, monoclonal anti-NeuN (1:200; MAB377, Merck, Burlington, MA, USA) and Alexa 594-conjugated goat anti-mouse (1:500; Life Technologies, Carlsbad, CA, USA) were used as primary and secondary antibodies, respectively. Sections were coverslipped and observed using an inverted fluorescence microscope (IX83 cellSense Dimension; Olympus, Tokyo, Japan) or inverted laser-scanning confocal microscope (LSM510 META; Carl Zeiss, Oberkochen, Germany). Images were captured as Z-stacks (8 z-sections, 0.5 μm apart).

ADAR2^flox/flox mice were injected with AAV-GFP (n = 10) or AAV-GFP/Cre (n = 10) into the NAc. Mouse brains were obtained 1 month later, and quickly frozen in liquid nitrogen. Coronal sections (15 μm thick) were cut using a cryostat and mounted onto membrane slides (PEN-membrane 2.0 μm; Leica Microsystems, Wetzlar, Germany). NAc samples from mounted coronal sections were obtained by 1-mm punch biopsy, followed by RT-PCR amplification using VolcanoCell2G 2 × RT-PCR Master Mix (myPLOS Biotec, Konstanz, Germany). PCR products were treated with ExoSAP-IT (Affymetrix, Santa Clara, CA, USA) and subjected to sequencing reaction (BigDye Terminator v1.1 Cycle Sequencing Kit, ThermoFisher, Waltham, MA, USA). Primer sequences are described in Table 1. Sequencing analysis was performed with an Applied Biosystems 3130 Genetic Analyzer (ThermoFisher, Waltham, MA, USA). DNA sequencing data were converted to waveform data, and the peaks of adenosine (A) and guanosine (G) were quantified using ImageJ software¹. RNA editing frequency was calculated as A/(A + G) × 100 (%).

ADAR2^flox/flox mice were injected with AAV-GFP (n = 55) or AAV-GFP/Cre (n = 66) into the NAc. Mice were subjected to anxiety- and depression-like behavior tests and ethanol consumption tests. The order of each testing session was: (1) anhedonia test; (2) light/dark transition test; (3) open field test; (4) elevated plus maze (EPM); (5) forced swimming test; (6) tail suspension test (TST). After chronic ethanol exposure; (7) ethanol-drinking behavior test; and (8) CPP test were performed. A home-cage activity test was performed using an independent cohort of mice. The test battery was performed at 1–3 day inter-test intervals. We checked correct microinjection of AAV into the bilateral NAc after behavioral tests.

Mice were allowed free access to water and food prior to the anhedonia test. During testing, mice were given free choice between two bottles, one with water and another with 1% sucrose solution, for 24 h (Shirahase et al., 2016). Mice were allowed free access to food during the test period. After 12 h, the positions of the bottles were switched to exclude possible effects of side preference in drinking behavior. The consumption (ml) of sucrose solution and water were estimated after 24 h. Sucrose consumption was calculated as sucrose intake (g) per sucrose volume (ml) per body weight (kg). Water intake and total intake were calculated as the volume of fluid intake (ml) per body weight (kg).

The light/dark transition test was performed as previously described (Aoki et al., 2016). The apparatus used for the light/dark transition test comprised a cage (14 × 14 cm) divided into two sections of equal size by a partition with a door (Melquest Co., Ltd, Toyama, Japan). One chamber was brightly illuminated (400 lux), whereas the other chamber was dark (2 lux). Mice were placed into the dark side and allowed to move freely between the two chambers with the door open for 10 min. The total number of transitions, time spent in each compartment, and latency of first movement to the light side were automatically recorded using an animal movement analysis system (SCANET-40; Melquest Co., Ltd).

Each subject was allowed to move freely in the open field box (60 × 60 cm) for 30 min. Time spent in the center area of the open field (30 × 30 cm) was measured using SMART v3.0 software (Panlab SL, Barcelona, Spain).

The EPM test was performed as described previously (Aoki et al., 2016). The EPM consisted of two open arms (30 × 5 cm) and two enclosed arms of the same size, with 13-cm-high transparent walls. The arms and central square were constructed from gray plastic sheets elevated to a height of 50 cm above the floor. To minimize the likelihood of animals falling from the apparatus, 5-mm-high Plexiglas sides were used for the open arms. Arms of the same type were arranged at opposite sides to each other. The device was set up under low illumination (center square, 300 lux). Each mouse was placed in a closed arm of the maze. Mouse behavior was recorded during a 10-min test period. The number of entries into and the time spent on open arms were recorded. For data analysis, the following two measures were used: percentage of entries into open arms and duration of stay on open arms. Data acquisition and analysis were performed using SMART v3.0 software.

The FST apparatus consisted of four Plexiglas cylinders. The cylinders were filled with water at a temperature of 23°C up to a height of 10 cm. Mice were placed into the cylinders and images recorded over a 10-min period. Data acquisition and analysis were performed using SMART v3.0 software. Immobility time was measured during 2–6 min of the test period (Aoki et al., 2016).

The TST was performed according to a previously described procedure (Watanabe et al., 2011). Mice were suspended 30 cm above the floor in a visually isolated area using adhesive tape placed 1 cm from the tip of the tail. Immobility duration was recorded over a 10-min test period. Images were captured using a USB camera (Webcam C270; Logicool, Tokyo, Japan). Data acquisition and analysis were performed using SMART v3.0 software.

Mice were placed in a home cage (22 × 22 × 18.5 cm) for 7 days prior to the home-cage activity test. Home-cage activity was recoded for 3 days using an animal movement analysis system (SCANET-40). Mice were maintained under a 12-h light/dark cycle (lights on from 08:00 to 20:00 h) with free access to water and food.

Chronically ethanol-exposed mice were produced as previously described (Yoshimoto et al., 2012). Briefly, mice were exposed to 22–27 mg/l of ethanol vapor during dark phase for 20 days using an intermittent 3–6 h/day schedule that mimics cyclical patterns of ethanol consumption. Blood ethanol concentration of mice was not measured. Mice were maintained in the ethanol vapor chamber under a 12-h light/dark cycle (lights on from 08:00 to 20:00 h) with free access to water and food.

After chronic ethanol exposure, mice were withdrawn from ethanol and maintained under normal experimental conditions for 4 h. For the ethanol-drinking behavior test, mice were provided with 10% (v/v) ethanol solution for 4 h, and their consumption was measured (Watanabe et al., 2014). The total amount of ethanol intake was represented as ethanol (g)/body weight (kg).

The CPP apparatus was divided into two sections of equal size (14 × 14 cm) by a partition with a door (Melquest Co. Ltd, Toyama, Japan). One box was painted black with a smooth floor and the other was painted white with a textured floor. The CPP test was performed 1–2 days after the ethanol drinking behavior test according to a previously described procedure (Thanos et al., 2005). All mice were allowed to recover under normal conditions for 3–5 days before the conditioning phase. In the preconditioning phase (days 1–3), each mouse was placed in the CPP apparatus for 30 min. Data from the 3rd day was analyzed for any unconditioned box preference. In the conditioning phase (days 4–11), mice were given saline in the black box on days 4, 6, 8, and 10. On days 5, 7, 9, and 11, the same mice were given ethanol (2 g/kg i.p. 20% (v/v) EtOH in saline) in the opposite chamber (for 30 min/trial). In the test phase (day 12), mice were placed in the apparatus and allowed free access to all boxes for 30 min. Each test session (days 3 and 12) was automatically recorded using an animal movement analysis system (SCANET-40).

Statistical analysis was performed using Stat View (SAS Institute, Cary, NC, USA). Significance of differences between two groups was calculated using Student’s t-tests. Ethanol-drinking behavior and CPP test data were analyzed by two-way analyses of variance (ANOVA; Bonferroni’s post hoc test). The alpha level was set to 0.05. Results of statistical analysis of RNA editing efficiency and behavioral tests were summarized in Table 2.

To generate NAc-specific ADAR2 knockout mice with a C57BL/6J genetic background, AAV-GFP/Cre was injected into the NAc (core and shell) of ADAR2^flox/flox mice (Figure 1A). Injection into the NAc showed about 500 μm diameter spread of AAV. Genomic PCR analysis showed that the recombination of ADAR2^flox/flox gene by Cre recombinase occurred in the NAc of AAV-GFP/Cre-injected mice (Figure 1B). In immunohistochemical analysis, ADAR2 expression was not detected in AAV-infected GFP/Cre-positive neurons in the NAc, whereas both ADAR2 and GFP expression were observed in the control AAV-GFP-infected neurons (Figure 1C). The loss of ADAR2 expression in AAV-GFP/Cre-injected NAc was not due to cell death, because numbers of NeuN-positive cells were not reduced and both NeuN- and GFP-positive neurons were observed in control AAV-GFP-injected NAc (Figure 2). Four weeks after the injection, we examined the influence of ADAR2-knockout on RNA editing. Sections from the NAc were lysed, and cDNAs were synthesized. The RNA editing frequency of ADAR2-dependent RNA editing sites, GluA2 Q/R, 5-HT_2CR site D and CYFIP2 K/E, was measured by direct sequencing analysis of cDNAs. Compared with that of AAV-GFP-injected mice, the RNA editing frequency of the three editing sites was significantly lower in the NAc of NAc-specific ADAR2 knockout mice (Figures 3A,B). The editing frequency of the ADAR2-mediated RNA editing sites 5-HT_2CR site D and CYFIP2 K/E site was reduced from 60% to 70% in controls to approximately 20% in ADAR2 knockout mice. Editing frequency of the GluA2 Q/R site was also lower in ADAR2 knockout mice (60%, compared with 100% in control mice). In contrast, the RNA editing frequency of all sites in the cortex was normal (Figures 3A,B). The body weight of control (GFP) and NAc-specific ADAR2 knockout mice (Cre) was measured 4 weeks after AAV injection. All mice appeared to be healthy, and mean body weight was not significantly different between the groups (Figure 3C).

We characterized the behavioral phenotypes of NAc-specific ADAR2 knockout mice 4 weeks after the AAV injection. Mice were subjected to a light/dark transition test, open field test and EPM test as analyses of anxiety-related behaviors. In the light/dark transition test, the total number of transitions between the light and dark chambers was significantly lower in NAc-specific ADAR2 knockout mice (p = 0.0069; Figure 4A), while there were no significant differences in the stay time in the light chamber or latency to first enter the light chamber (Figures 4B,C). In the open field test, no significant difference between the two groups was observed in the time spent in the central area (Figures 4D,E). However, the distance traveled by NAc-specific ADAR2 knockout mice was significantly lower than that of control mice (p = 0.0058; Figure 4F). In the EPM test, major differences were not seen in number and percentage of entries into the open arms, percentage of stay time in the open arm or the distance traveled (Figures 4G–J). A diurnal rhythm of spontaneous locomotor activity was recorded for 3 days. Both NAc-specific ADAR2 knockout mice and control mice showed normal rhythms and similar gross levels of spontaneous locomotor activity (Figure 5).

Next, we examined despair-related behaviors using the FST and TST. The results of the FST revealed no significant difference in immobility time between control and NAc-specific ADAR2 knockout mice (Figure 6A). Similarly, there was no significant difference in immobility time in the TST (Figure 6B). Mice were subjected to the sucrose preference test as a measure of anhedonia-related behavior. Mice had the choice of either drinking 1% sucrose solution or water for 24 h. There were no differences in the consumption of sucrose solution or water between the two groups (Figures 6C–E). These results showed that despair- and anhedonia-related phenotypes of NAc-specific ADAR2 knockout mice were normal.

GFP and Cre/GFP mice were exposed to ethanol vapor for 20 days prior to ethanol-drinking behavior and ethanol preference tests. Chronic ethanol vapor exposure significantly increased 10% ethanol intake in GFP mice (genotype × treatment: F_(1,35) = 4.911, p = 0.0333, two-way ANOVA; GFP-control vs. GFP-ethanol vapor: p = 0.0009, Bonferroni’s post hoc test), while Cre/GFP mice did not show enhanced ethanol intake (Figure 7A). This result suggests that ADAR2-mediated RNA editing in the NAc is involved in ethanol-drinking behavior. Following the ethanol-drinking behavior test, ethanol preference was measured in these mice using the ethanol CPP test. In control mice, there was no significant difference in CPP score between GFP and Cre/GFP mice, and the mice in both cases did not develop ethanol CPP (Figure 7B). In contrast, there was a significant difference in the CPP scores of GFP mice after chronic ethanol exposure between the pre-conditioning and conditioning phases (Figure 7C; ethanol-conditioned: F_(1,34) = 7.194, p = 0.0112, two-way ANOVA; p = 0.0222 post hoc test), whereas there was no significant difference in the CPP scores of Cre/GFP mice after chronic ethanol exposure between the pre-conditioning and conditioning phases (Figure 7C). These results indicate that ADAR2-mediated RNA editing in the NAc is involved in the enhancement of ethanol preference induced by chronic ethanol exposure.