Wild-type C57BL/6J mice (male, 8-10 weeks old) were used for validation of behavioral experiments. For DREADD, optogenetic, and calcium imaging experiments, male heterozygous D1-Cre (FK150Gsat) and D2-Cre (ER44Gsat) mice were used (DREADD, D1-Cre, n = 12, one mouse was excluded due to no viral expression; D2-Cre, n = 12, one mouse was excluded due to unstable behavior; optogenetics, D2-Cre, n = 19, 2 mice were excluded due to insufficient conditioning; calcium imaging, D1-Cre, n = 3; D2-Cre, n = 4, one mouse was excluded because of incorrect GRIN lens placement). D1-Cre and D2-Cre were maintained in a C57BL/6J background. Animals were housed on a 12-hour light/dark cycle. Behavioral studies were conducted during the light cycle. Mice were kept on water restriction during behavioral testing. For all behavioral experiments except for calcium imaging experiment, mice were grouped housed throughout the experiments. For calcium imaging experiments, mice were singly housed after GRIN lens implantation. All experiments conformed to the guidelines of the National Institutes of Health experimental procedures, and were approved by the Animal Experimental Committee of Institute for Protein Research at Osaka University (approval ID 29-02-1).
All mice used in this study were anesthetized with ketamine (100 mg/kg) and xylazine (20 mg/kg) for surgical procedures and placed in a stereotaxic frame (Kopf Instruments).
For DREADD experiments, heterozygous D1-Cre or D2-Cre mice were bilaterally injected with 400 nl of AAV5-hSyn-DIO-hM4Di(Gi)-mCherry (5.1×1012 GC/ml, UNC) or AAV5-hSyn-DIO-mCherry (5.2×1012 GC/ml, UNC) using a Nanoject II instrument (Drummond) at a rate of 100 nl/min (coordinates in mm: AP +1.20, ML ±1.25 from bregma, and DV −3.50 from brain surface. The injection pipette remained in place for 5–10 min to reduce backflow.
For optogenetics experiments, heterozygous D2-Cre mice were bilaterally injected with 400 nl of AAV5-CAG-FLEX-ArchT3.0-tdTomato (1.3×1013 GC/ml, Addgene) or AAV5-EF1a-DIO-eYFP (1.3×1013 GC/ml, Addgene) were using a Nanoject III instrument (Drummond) at a rate of 100 nl/min (coordinates in mm: AP +1.20, ML ±1.25 from bregma, and DV −3.50 from brain surface. The injection pipette remained in place for 5–10 min to reduce backflow. After retraction, 200 µm diameter (NA 0.37) optic fibers (Thorlabs) were bilaterally implanted and fixed in place with the dental cement (Superbond) at AP +1.20, ML ±1.30 from bregma, and DV −3.20 from brain surface.
For calcium imaging experiments, heterozygous D1-Cre or D2-Cre mice were unilaterally injected with 1200 nl of AAV9-FLEX-jGCaMP7f (9.6×1012 GC/ml, Addgene) were stereotaxically injected using a Nanoject III instrument (Drummond) at a rate of 100 nl/min (coordinates in mm: AP +1.20, ML ±1.25 from bregma, and DV −3.60 and −3.10 from brain surface. The injection pipette remained in place for 5–10 min to reduce backflow. After virus injection, a sterile 21-gauge needle was slowly lowered into the brain to a depth of -2.0 mm from the brain surface to aspirate brain tissue above the NAc. A GRIN lens (600 µm diameter, Inscopix) was slowly lowered into the brain to a depth of -3.20 mm from the brain surface by using a GRIN lens holder (Inscopix). We secured the GRIN lens to the skull with dental cement (Superbond). A silicone elastomer (Kwik-Cast; World Precision Instruments) was applied to the top of the lens to prevent external damage. Four-to-six weeks after lens implantation, a baseplate (Inscopix) attached to the miniature microscope (nVista; Inscopix) was positioned above the GRIN lens. The focal plane was adjusted until blood vessels could be clearly observed. After adjustment, the baseplate was secured in place with the dental cement.
Apparatus. Training and testing were conducted in a Bussey-Saksida touchscreen chamber (Lafayette Instrument). A black plastic mask with 2 windows (70×75 mm2 spaced, 5 mm apart, 16 mm above the floor) was placed in front of the touchscreen. ABET II and WhiskerServer software (Lafayette) were used to control operant system and data collection.
Pretraining. As the first phase (3 days), mice were habituated to the chamber in 40-min sessions. Diluted condensed milk (7 µl, Morinaga Milk) was dispensed in the reward magazine every 10 sec. In the following phase (1 day), a stimulus was randomly displayed in 1 of the 2 windows. After a 30-sec stimulus presentation, the milk reward (20 µl) was delivered with a tone (3 kHz) and the inside of the magazine was illuminated. When mice collected the reward, the magazine light went out, and the next trial commenced (60 trials, or up to 60 min) with a new stimulus after a 20-sec intertrial interval (ITI). In the next phase, stimuli were randomly displayed in one of 2 windows, and mice were obligated to touch the stimulus to receive a reward. In the final phase of the pretraining, when a blank window was touched, mice were punished with a 5-sec time-out. After reaching criterion (77% correct for 2 consecutive days), mice moved on to basic training.
Basic training. Mice were tested 5–6 days per week (60 trials per day, or up to 60 min). Each trial was initiated after mice nose-poked in the magazine. Visual cues were presented until mice responded at either window.
For the VD-Attend task, two visual cues (marble and a random image) were presented in the touchscreen. The random image was pseudorandomly chosen from 51 images. If the mouse responded to the correct (marble) visual cue, a milk reward (7 µl) was delivered with a tone (3 kHz) and the magazine was illuminated. When mice collected the reward, the magazine light went out, and the next trial commenced (60 trials, or up to 60 min) with a new stimulus after a 20-sec intertrial interval (ITI). If the mouse responded to the incorrect (random) visual cue, the mouse was punished with a 5-sec time-out (house light on).
For the VD-Avoid task, two visual cues (flag and a random image) were presented in the touchscreen. If the mouse responded to the correct (random) visual cue, a milk reward (7 µl) was delivered with a tone (3 kHz) and the magazine was illuminated. If the mouse responded to the incorrect (flag) visual cue, the mouse was punished with a 5-sec time-out (house light on).
A response at a random image was recorded as a correct response, while a response to visual cue “Flag” was recorded as an incorrect response.
After reaching criterion (>80% correct for 2 consecutive days), mice moved on to the test phase for DREADD experiments or cable habituation for optogenetic experiments, respectively.
For DREADD experiments, vehicle on days 1 and 3, or CNO (1.0 mg/kg diluted with vehicle, Sigma Aldrich) on days 2 and 4, was intraperitoneally administered 30 min before the session.
For optogenetic inhibition experiments, once the performance stabilized (>80% correct for 2 consecutive days) with the fiber optic cables attatched, optogenetic stimulation experiments were commences. LED power was set to 1-3 mW. Stimulation schedule was counterbalanced.
For calcium imaging experiments, data was acquired at 20 Hz with 0.6 mW LED at the first session of the basic training (Novice) and the session after reaching criterion (the criterion session; Expert). After acquisition, calcium recording files were temporally (factor of 2) and spatially (factor of 4) downsampled and motion-corrected using Inscopix Data Processing software ver 1.3.0. The fluorescent traces of individual neurons were extracted from these images by CNMFe 30. Z-scores were calculated from all recording data. The first three principal components (PCs) of the Z-scores of all neurons in the correct and error trials were calculated using principal component analysis (PCA), with the singular value decomposition algorithm. Hierarchical clustering of the first three PCs was then performed using a Euclidean distance metric and a complete agglomeration method.
Percentage correct (correct trials divided by correct plus incorrect trials, recorded as percent), and latencies to correct response, incorrect response, and reward collection were monitored in all behavioral experiments.
Animals were deeply anesthetized and transcardially perfused with 0.01 M PBS followed by 4% paraformaldehyde (PFA) in 0.1 M PB (pH 7.4). Brains were removed and post-fixed with 4% PFA at 4 ºC for 2 days. After cryoprotection, brains were embedded in OCT compound and cryosectioned (40 µm). Sections were mounted with antifade mouting medium with DAPI (Vectashield). Stitched images were acquired using a Keyence BZ-X800 microscope.
Prism (Graphpad) software was used for statistical analyses. The behavioral performances in wild-type were analyzed using unpaired t-test. DREADD data were analyzed using two-way RM ANOVA with Group (hM4Di, mCherry) and Drug Treatment (vehicle, CNO) or one-way ANOVA (D1-hM4Di, D2-hM4Di, D1/D2-mCherry). Optogenetic data were analyzed using two-way RM ANOVA with Group (ArchT, eYFP) and Light stimulation (OFF, ON) or History (After Correct, After Error) and Light stimulation (OFF, ON). Post hoc Sidak’s multiple comparisons test was performed when F-ratios were significant (p < 0.05). Comparisons of proportion of cells were made using chi-squared test. Simple linear regression was made to calculate the correlation of auROC between novice and expert. All data are expressed as means ± SEM.