A Male Mouse Model of WIN 55,212–2 Self-Administration to Study Cannabinoid Addiction
用于研究大麻素成瘾的雄性小鼠WIN 55,212–2自给药模型
发布: 2026年04月05日第16卷第7期 DOI: 10.21769/BioProtoc.5652 浏览次数: 419
评审: Shubham GargFereshteh AzediAnonymous reviewer(s)
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参见作者原研究论文
The authors used this protocol in:
Mar 2023
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Abstract
Despite substantial progress in preclinical cannabinoid research, translational studies on cannabis use disorders (CUD) are still insufficient due to the absence of robust, validated animal models that fully recapitulate the multifactorial clinical phenotype of human CUD. The complex nature of CUD and the incomplete understanding of its underlying neurobiological mechanisms contribute to the limited availability of effective treatments. To address this gap, we developed an operant conditioning–based mouse model that enables the identification of individual vulnerability or resilience to CUD development. This highly translational model is based on the Diagnostic and Statistical Manual of Mental Disorders, 5th Edition (DSM-5) criteria for substance use disorders. The model allows the assessment of addiction-like behaviors by evaluating three behavioral domains: 1) persistence of responding during periods of cannabinoid unavailability, 2) motivation for cannabinoid seeking measured using a progressive ratio schedule, and 3) compulsivity, assessed when cannabinoid reward is paired with an aversive consequence such as a mild electric foot shock. A major strength of this paradigm is its ability to quantify two phenotypic traits proposed as predisposing factors for addiction vulnerability and two parameters related to craving. In addition, the model is specifically designed to evaluate genetic and circuit-level manipulations using chemogenetic approaches, with minor modifications required by surgical viral-vector delivery. Using this protocol, we can determine whether altering the excitability of specific neural networks promotes resilience or vulnerability to developing cannabinoid addiction. Elucidating these mechanisms is expected to facilitate the identification of novel and more effective therapeutic interventions for CUD.
Key features
• Operant conditioning–based mouse model to study cannabis use disorders (CUD) based on DSM-5 substance use disorder criteria.
• Enables assessment of addiction-like behaviors across persistence, motivation (progressive ratio), and compulsivity under punishment, allowing stratification of vulnerable versus resilient individuals.
• Quantifies phenotypic traits linked to cannabinoid addiction vulnerability and behavioral signatures associated with craving for cannabinoids.
• Compatible with genetic and circuit-level manipulations to test how specific neural networks modulate CUD-related behaviors.
Keywords: Cannabis use disorder (大麻使用障碍)
Vulnerability (易感性)
Resilience (抗逆性)
Operant conditioning (操作性条件反射)
Chemogenetics (化学遗传学)
Viral vector approach (病毒载体方法)
Compulsivity (强迫性行为)
Impulsivity (冲动性)
Motivation (动机)
Addiction-like behavior (类成瘾行为)
Graphical overview
Graphical abstract of the cannabinoid addiction mouse model
Background
Cannabinoids remain the most commonly used illicit drugs worldwide, with consumption increasing in recent years. In Europe, an estimated 8.0%–8.3% of adults aged 15–64 used cannabis in the last year [1]. An increasing perception that cannabinoids are relatively safe has contributed to more permissive attitudes toward use, paralleling rising rates of cannabis use disorder (CUD) [1,2].
CUD is defined as a chronically relapsing neuropsychiatric disorder, diagnosed using the Diagnostic and Statistical Manual of Mental Disorders, 5th edition (DSM-5) criteria, where addiction is equivalent to severe substance use disorder (SUD), requiring fulfillment of at least six of eleven criteria [3]. Cannabis addiction is a complex, multifactorial disorder that emerges from interactions between genetic susceptibility and environmental influences, including epigenetic regulation [4,5]. Not all individuals with repeated exposure transition to addiction, highlighting interindividual variability in vulnerability and resilience [6]. Although numerous cannabinoid-induced neuroadaptations have been described, the mechanisms that specifically confer vulnerability remain incompletely understood, and valid animal models of cannabinoid addiction are still lacking.
Rodent models of operant self-administration have been widely used to study cannabinoid-related behaviors [7–13]. These paradigms have advanced understanding of reward and reinforcement but typically rely on a single behavioral signature and therefore do not capture the multidimensional diagnostic construct of CUD. Moreover, robust self-administration and escalation are more consistently observed with synthetic cannabinoids than with Δ9-tetrahydrocannabinol (THC), whose lower reinforcing efficacy and aversive effects at high doses complicate modeling of cannabis addiction.
The operant conditioning–based protocol described here addresses these limitations by providing a flexible, translational framework to assess addiction-like behaviors induced by synthetic cannabinoids [7–12]. The procedure is aligned with DSM-5 substance use disorder criteria and interrogates three key behavioral domains within the same subjects: (i) persistence of responding during drug unavailability, (ii) motivation to obtain the drug using a progressive ratio schedule, and (iii) compulsive drug seeking when access is paired with an aversive consequence. This design enables longitudinal tracking of addiction-like trajectories at the individual level [7,8].
A major strength of the protocol is its sensitivity to individual differences, allowing classification of animals as vulnerable or resilient based on composite behavioral scores rather than group averages. The task is compatible with viral vector–based approaches and is particularly suited to circuit-specific manipulations, facilitating rapid evaluation of how defined neural networks modulate cannabinoid seeking. Limitations include the need for operant training, specialized equipment, and the potential confounding influence of stress or pain sensitivity on punishment-based measures. Beyond CUD, the framework can be adapted to other drugs of abuse, polysubstance paradigms, developmental exposures, sex differences, and gene–environment interactions, providing a robust platform to dissect the behavioral and circuit-level mechanisms underlying cannabinoid addiction.
Materials and reagents
Biological materials
1. 8–10-week-old C57BL/6J male mice (Charles River, France, or any company that provides mice for laboratories)
Reagents
1. Distilled water
2. Ethanol 70%
3. Iodine, 500 mL (Betadine, MEDA Pharma S.A.U., catalog number: 716720)
4. Physiological saline (0.9%, 250 mL) (Laboratorios ERN, catalog number: 999790.8)
5. Glucose serum (GlucosaVet 5 g/100 mL) (B. Braun Vet Care, catalog number: 1248 ESP)
6. Sodium heparin (Heparina Hospira 5%) (Pfizer, catalog number: 654753.3)
7. Ophthalmic ointment (Xilin night, 5 g) (Visufarma, catalog number: 2919-PS-CM)
8. Blastoestimulina (1%, 30 g) (Almirall, catalog number: 719385)
9. VirkonTM S (Laboratorios Zotal, catalog number: 0065-P)
10. Vetflurane (Isoflurane, 250 mL) (Virbac, catalog number: 2199-ESP)
11. Clozapine N-oxide (CNO, 25 mg) (Enzo Life Sciences, catalog number: BML-NS105-0025); diluted in 0.9% sterile saline (5 mg/mL).
Note: CNO powder is stored at room temperature, protected from light. Stock solutions are prepared in saline, aliquoted, and stored at 4 °C. Working solutions are freshly prepared on the day of injection. Shelf-life: typically stable for 2 years after opening.
12. Anesthesia reagents
a. Ketamine hydrochloride (75 mg/kg of body weight, 10 mL) (Ketamidor) (Richter Pharma AG, catalog number: 580393); dissolved in sterile 0.9% physiological saline
b. Medetomidine hydrochloride (1 mg/kg of body weight) (Domtor) (Esteve, catalog number: 570686); dissolved in sterile 0.9% physiological saline
c. Atipamezole hydrochloride (2.5 mg/kg of body weight) (Revertor) (Virbac, catalog number: 570559); dissolved in sterile 0.9% physiological saline
d. Gentamicine (1 mg/kg of body weight) (Genta-Gobens) (Laboratorios Normon, catalog number: 999037); dissolved in sterile 0.9% physiological saline
e. Meloxicam (2 mg/kg of body weight) (Metacam) (Boehringer Ingelheim, catalog number: 059/02/08CVFPT); dissolved in sterile 0.9% physiological saline
13. Thiopental (Tiobarbital, 0.5 g) (B. Braun)
14. Antibiotic ointment (Bactroban, GlaxoSmithKline)
15. Viral vectors
a. AAV8-hSyn-DIO-hM4D(Gi)-mCherry (1.21 × 1013 gc/mL) (Viral Vector Production Unit of Universitat Autònoma de Barcelona)
b. AAV8-hSyn-DIO-mCherry (1.19 × 1013 gc/mL) (Viral Vector Production Unit of Universitat Autònoma de Barcelona)
c. AAVrg pmSyn1-EBFP-Cre (6 × 1012 vg/mL) (Addgene, catalog number: 51507-AAVrg)
Note: Viral vectors are stored at –80 °C upon arrival and aliquoted to avoid repeated freeze–thaw cycles. Vials are kept on ice during surgical procedures and used within the manufacturer’s recommended stability period. Shelf-life: typically stable for 2 years at -80 °C.
16. WIN 55,212-2 [(R)-(+)-WIN 55,212-2 mesylate salt] (Sigma-Aldrich, catalog number: W102
17. Tween 80 (Sigma-Aldrich, catalog number: P8074)
18. 4% paraformaldehyde (PFA) in 0.1 M Na2HPO4/0.1 M NaH2PO4 buffer (PB) (Sigma-Aldrich, catalog number: P6148)
19. Sucrose (Sigma-Aldrich, catalog number: S7903)
Solutions
1. Heparin 0.0003 mg/mL (see Recipes)
2. Ketamine (7.5 mg/mL) + Medetomidine (0.2 mg/mL) (see Recipes)
3. Atipamezole 1 mg/mL (see Recipes)
4. Gentamicin 0.3 mg/mL (see Recipes)
5. Meloxicam 0.5 mg/mL (see Recipes)
6. WIN 55,212-2 (see Recipes)
Recipes
1. Heparin 0.0003 mg/mL
0.12 mL of sodium heparin + 20 mL of sterile 0.9% physiological saline.
Note: Store heparin at 4 °C in sterile stock vials and prepare fresh working solutions on the day of use to ensure stability and sterility.
2. Ketamine (7.5 mg/mL) + Medetomidine (0.2 mg/mL)
0.75 mL of ketamine hydrochloride (100 mg/mL Ketamidor) + 1 mL of medetomidine hydrochloride (1 mg/mL, Dormitor) + 8.25 mL of sterile 0.9% physiological saline.
3. Atipamezole 1 mg/mL
2 mL of atipamezole hydrochloride (2.5 mg/kg of body weight, Revertor) + 8 mL of sterile 0.9% physiological saline.
4. Gentamicin 0.3 mg/mL
0.06 mL of gentamicin (40 mg/mL, Genta-Gobens) + 10 mL of 0.9% physiological saline.
5. Meloxicam 0.5 mg/mL
0.5 mL of meloxicam (40 mg/mL, Metacam) + 40 mL of glucose serum (GlucosaVet 5 g/100 mL).
6. WIN 55,212-2
Dissolve WIN 55,212-2 powder in one drop of Tween 80 and store at room temperature, protected from light. Prepare stock solutions in saline, aliquot, and store at 4 °C. Prepare working solutions freshly on the day of injection and self-administration session. Shelf-life: typically stable for 2 years.
Laboratory Supplies
1. Scalpel (Agnthos, catalog number: 02-036-04)
2. Manual drill (Plastics One, catalog number: DH-1)
3. Blunt-tipped surgical scissors (Agnthos, catalog number: 03-022-105)
4. Curved iris forceps (Agnthos, catalog number: 08-513-005)
5. Suture clips (Agnthos, catalog number: 08-922-125)
6. Surgical clips (Agnthos, catalog number: 22-620-007)
7. Fine scissors 105 mm-4¼ (Allgaier Instrumente, catalog number: 03-320-105)
8. Curved forceps (Allgaier Instrumente, catalog number: 08-421-100)
9. Bishop-Harman forceps serrated 0.5 mm (Agnthos, catalog number: 11069-08)
10. Graefe forceps straight serrated 0.8 mm (Agnthos, catalog number: 11050-10)
11. 2× Dumont 5 forceps (Fine Science Tools, catalog number: 11251-10)
12. Rolled cotton 100% (Acofarma, Acofar®, catalog number: 4957051)
13. Weighing scale
14. Tygon tubing (0.508 × 1.524 mm) (Tecny fluor, catalog number: S-54-HL)
15. Catheter for mouse (mouse jugular catheter) (Sai Infusions, model: MJC-01)
16. Microsyringe (10 μL, model 1701 N S.Y.R., cemented N.D.L., 26 ga, 2 in, point style 3) (Hamilton company, catalog number: 80039)
17. Polyethylene tubing (PE-20, 0.381 × 1.092 mm) (Plastics One, catalog number: PT-C315CT)
18. Bilateral guide cannula (26-gauge cannula cut 12 mm below pedestal) (Plastics One, catalog number: C235GS-5/Spc)
19. Bilateral internal cannula (33-gauge internal cannula fits 12 mm with 3 mm projection) (Plastics One, catalog number: C235GS-5/Spc)
20. Osmotic minipumps (flow rate of 0.25 μL/h for 28 days) (ALZET, model: 2004, catalog number: 0000298)
21. Suture thread (black braided silk, TB10, 3/8 TRIANG 15 mm 4/0 90 cm) (LorcaMarín, catalog number: 55327-50U)
22. 20 gauge 1½ inch needles (BD Microlance 3, catalog number: 301300)
Equipment
1. Mouse operant self-administration chambers (Med Associates, model: ENV-307A-CT)
Operant chambers are equipped with two nose-poke holes (holes of 1.2 cm diameter), one randomly selected as the active nose poke and the other as the inactive. Nose-poking on the active nose poke results in an infusion of the synthetic cannabinoid agonist WIN 55,212-2 paired with a stimulus-light (2 s, associated-cue) (Med Associates, catalog number: ENV-321M), located above the active nose poke. Nose-poking on the inactive hole has no consequences. A house light is located on the ceiling of each chamber (Med Associates, catalog number: ENV-315M), and two cue lights are positioned at the active nose poke, one inside the hole and one above it (Figure 1). Nose pokes at the active hole trigger a WIN 55,212–2 infusion according to the programmed schedule, coincident with illumination of the cue light above the hole, whereas responses at the inactive hole have no programmed consequences. Chambers consist of aluminum and acrylic walls, enclosed within sound- and light-attenuating cubicles, equipped with ventilating fans that also provide constant white noise at 55–65 dBA. The floor consists of parallel metal bars that deliver foot shocks (0.18 mA) during the punishment test for compulsive-like behavior (Med Associates, catalog number: ENV-307A-GF). The synthetic full agonist cannabinoid WIN 55,212–2 (12.5 μg/kg/infusion) is infused in a volume of 23.5 μL over 2 s via a syringe mounted on a microinfusion pump (Med-Associates, model: PHM-100A), connected through flexible polymer Tygon tubing (0.96 mm outer diameter) (Tecny fluor) to a single-channel liquid swivel (Polysulfone 22 GA single-channel disposable swivel 610014; Harvard apparatus/Panlab) and the mouse intravenous catheter (Sai Infusions, U.S.A). Although Med Associates equipment was used in the present study, functionally equivalent operant conditioning systems and programmable infusion pumps from other manufacturers may be used if they allow precise control of stimulus presentation, response detection, and infusion parameters.
Figure 1. Operant self-administration chamber. (A) Diagram of the operant chamber. The operant chamber is equipped with two nose pokes (active and inactive), a stimulus light, and an infusion pump. The chamber floor is a grid that delivers electric foot shocks during the shock test session. (B) General view of the operant box with the box light, the active nose poke, the cue-light, the grid floor that delivers electric foot shocks, and the infusion pump for drug delivery.
2. Stereotaxic apparatus (100 µm resolution) (Koft instruments, model: 900)
3. Standing magnifier (Carl Zeiss, model: OPMI 1 FR)
4. Microinfusion pump (P.H.D. 2000, Harvard Apparatus, catalog number: MA1 70-20xx)
5. Animal trimmer (Artero, catalog number: M630)
6. Cold light (Leica C.L.S. 150×, Leica Microsystems)
7. Heating pad (60 W, 50 Hz) (Daga, catalog number: N2P 220-230)
8. Hot bead sterilizer (FST 250) (Agnthos, catalog number: 18000-45)
9. Activity boxes (10.8 × 20.3 × 18.6 cm) (Imetronic, Pessac, France)
Software and datasets
1. Med-PC 5 software (Med Associates Inc., U.S.A.) to record all operant behavior in the self-administration chambers
Procedure
文章信息
稿件历史记录
提交日期: Dec 30, 2025
接收日期: Mar 3, 2026
在线发布日期: Mar 13, 2026
出版日期: Apr 5, 2026
版权信息
© 2026 The Author(s); This is an open access article under the CC BY-NC license (https://creativecommons.org/licenses/by-nc/4.0/).
如何引用
Martín-García, E., Ponce-Beti, M. F., Gusinskaia, T., López-Moraga, A., Capellán, R. and Maldonado, R. (2026). A Male Mouse Model of WIN 55,212–2 Self-Administration to Study Cannabinoid Addiction. Bio-protocol 16(7): e5652. DOI: 10.21769/BioProtoc.5652.
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分类
神经科学 > 行为神经科学 > 成瘾
神经科学 > 行为神经科学 > 实验动物模型
神经科学 > 基础技术 > 化学遗传学
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