Materials and Reagents

Materials

1. Chocolate-flavored pellets (20 mg/pellet, 5TUL #1811223, TestDiet, Richmond, IN, USA)
2. Microsyringe (10 μl, Model 1701 N S.Y.R., Cemented N.D.L., 26 ga, 2 in, point style 3, #80039, Hamilton company, N.V., U.S.A.)
3. Polyethylene tubing (PE-20, #C315CT , Plastics One, U.K.)
4. Bilateral guide cannula (26-gauge cannula cut 12 mm below pedestal, #C235GS-5/Spc, Plastics One, U.K.)
5. Bilateral internal cannula (33-gauge internal cannula fits 12 mm C235GS-5/Spc with 3 mm projection, Plastics One, U.K.)
6. Osmotic minipumps (flow rate of 0.25 μl/h for 28 days, Model 2004, #0000298 , Alzet, CA, U.S.A.)
7. Scalpel ( #02-036-040 , AgnTho's, Sweden)
8. Manual drill ( DH-1 , Plastics One, U.K.)
9. Blunt-tipped surgical scissors ( #03-022-105 , AgnTho's, Sweden)
10. Curved iris forceps ( #08-513-005 , AgnTho's, Sweden)
11. Suture clips ( #08-922-125 , AgnTho's, Sweden)
12. Surgical clips ( #22-620-007 , AgnTho's, Sweden)
13. Suture thread (black braided silk, TB10, 3/8 TRIANG 15 mm 4/0 90 cm, #55327-50U , LorcaMarín, Spain)
    

1. Distilled water
2. Ethanol 70%
3. Iodine (Betadine, 500 ml, #716720 , MEDA Pharma S.A.U., Spain)
4. Physiological saline (0.9%, 250 ml, #999790.8 , Laboratorios ERN, Spain)
5. Glucose serum (GlucosaVet 5g/100ml, #1248 ESP , B. Braun Vet Care, Spain)
6. Xilin night (5 g, #2919-PS-CM , Visufarma, Spain)
7. Blastoestimulina (1%, 30g, # 719385 , Almirall, Spain)
8. Vetflurane (Isoflurane, 250 ml, #2199-ESP , Virbac, Spain)
9. Clozapine N-oxide (CNO, 25 mg, #BML-NS105-0025 , Enzo Life Sciences, NY) diluted in 0.9% sterile saline (5 mg/ml)
10. Anesthesia reagents
    
    1. Ketamine hydrochloride (75 mg/kg of body weight, 10 ml, Ketamidor, #580393 , Richterpharma ag, Austria) dissolved in sterile 0.9% physiological saline
    2. Medetomidine hydrochloride (1 mg/kg of body weight, #570686 , Domtor; Esteve, Spain) dissolved in sterile 0.9% physiological saline
    3. Atipamezole hydrochloride (2.5 mg/kg of body weight, #570559 , Revertor; Virbac, Spain) dissolved in sterile 0.9% physiological saline
    4. Gentamicine (1 mg/kg of body weight, #999037 , Genta-Gobens; Laboratorios Normon, Spain) dissolved in sterile 0.9% physiological saline
    5. Meloxicam (2 mg/kg of body weight, Metacam; #059/02/08CVFPT , Boehringer Ingelheim, Rhein) dissolved in sterile 0.9% physiological saline
       
11. Viral vectors (storage at -80 °C). Examples
    
    1. AAV8-hSyn-DIO-hM4D(Gi)-mCherry (1.21E + 13 gc/ml, Viral Vector Production Unit of Universitat Autònoma de Barcelona)
    2. AAV8-hSyn-DIO-mCherry (1.19E + 13 gc/ml, Viral Vector Production Unit of Universitat Autònoma de Barcelona)
    3. AAVrg pmSyn1-EBFP-Cre (6 x 10¹² vg/ml, Addgene, viral prep # 51507-AAVrg )

Equipment

1. Mouse operant self-administration chambers (Model ENV-307A-CT , Med Associates, Georgia, VT, U.S.A.)
   The operant chambers are equipped with two retractable levers (#ENV-312-2M, Med Associates), one randomly selected as the active lever and the other as the inactive. Pressing on the active lever results in a food pellet delivery paired with a stimulus-light (associated-cue, #ENV-321M, Med Associates), located above the active lever, and while pressing on the inactive lever has no consequences. A food dispenser (#ENV-303M pellet receptacle, #ENV-203M-20, modular pellet dispenser, Med Associates) equidistant between the two levers permit the delivery of food pellets when required. The floor of the chambers is a grid floor (#ENV-307A-GF, Med Associates) that serves to deliver electric foot shocks in the session of shock test and serves as a contextual cue in the session of shock-induced suppression the day after the shock test. During the rest of the self-administration sessions, a metal sheet with holes is placed above the grid floor. Thus, mice can discriminate between different contexts. A house light is placed on the ceiling of the chamber (#ENV-315M, Med Associates). The chambers are made of aluminum and acrylic and are housed in sound- and light-attenuated boxes equipped with fans to provide ventilation and white noise. (Figure 1).
   
   
   Figure 1. Diagram and images of an operant self-administration chamber. A. Diagram of the operant chamber. The operant chamber is equipped with two retractable levers (active and inactive), a stimulus-light, and a food pellet dispenser. The floor of the chamber is a grid floor that delivers electric foot shocks in the session of shock test but is covered during the rest of self-administration sessions with a metal sheet with holes. B. Image with detail of the panel containing the active lever signaled by the cue-light and the metal sheet with holes. C. General view of the Skinner Box with the operant box light, the active lever, the cue-light, the grid floor that delivers electric foot shocks, the metal sheet with holes, and the food dispenser.


2. Stereotaxic apparatus (100-micron resolution, Model 900 , Koft instruments, C. A., U.S.A.)
3. Standing magnifier (OPMI 1 FR, Carl Zeiss, U.S. A.)
4. Microinfusion pump (P.H.D. 2000, #MA1 70-20xx , Harvard Apparatus, Holliston, MA, U.S.A.)
5. Animal trimmer ( #M630 , Artero, Spain)
6. Cold light (Leica C.L.S. 150x, Leica Microsystems, Spain)
7. Heating pad ( #N2P 220-230 , 60W, 50Hz, Daga, Spain)
8. Hot bead sterilizer (FST 250, #18000-45 , AgnTho's, Sweden)
   

Software

1. Med-PC Software (Med Associates Inc, U.S.A.). Software that registers all the behavior in the operant self-administration chambers
2. GraphPad Prism software (GraphPad Software, U.S.A.) to perform all graphs
3. SPSS software (I.B.M., version 25) to perform statistical data analysis
   

Procedure