Assessment of Substance Abuse Liability in Rodents: Self‐Administration, Drug Discrimination, and Locomotor Sensitization

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Abstract
Table of Contents
Materials
Figures
Literature Cited

Abstract

Assessing abuse liability is a crucial step in the development of a novel chemical entity (NCE) with central nervous system (CNS) activity or with chemical or pharmacological properties in common with known abused substances. Rodent assessment of abuse liability is highly attractive due to its relatively low cost and high predictive validity. Described in this unit are three rodent assays commonly used to provide data on the potential for abuse liability based on the acute effects of NCEs: specifically, self?administration, drug discrimination, and locomotor sensitization. As these assays provide insight into the potential abuse liability of NCEs as well as in vivo pharmacological mechanism(s) of action, they should form a key part of the development process for novel therapeutics aimed at treating CNS disorders. Curr. Protoc. Pharmacol. 58:5.62.1?5.62.22. © 2012 by John Wiley & Sons, Inc.

Keywords: abuse liability; self?administration; drug discrimination; locomotor sensitization; rat; mouse

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Introduction
Basic Protocol 1: Drug Self‐Administration in Rats
Alternate Protocol 1: Self‐Administration in Drug‐Naïve Rats
Basic Protocol 2: Drug Discrimination in Rats
Basic Protocol 3: Locomotor Sensitization
Reagents and Solutions
Commentary
Literature Cited
Figures

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Materials

Basic Protocol 1: Drug Self‐Administration in Rats

Materials

Rats: male Wistar or Sprague‐Dawley rats, 300 to 324 g upon arrival
Standard rat chow
Standard rat bedding
70% ethanol
Dustless purified precision pellets (45 mg) for food‐shaping (Bio‐Serv, cat. no. F0165; http://www.bio‐serv.com/)
Catheter flushing solution (see recipe )
Isoflurane
Oxygen
Saline: 0.9% (w/v) NaCl
Swabs (povidone‐iodine and alcohol)
Tissue glue
Topical antibiotic ointment
Carprofen solution (see recipe )
Drug solutions:
- Nicotine injection solution (see recipe )
- Cocaine injection solution (see recipe )
- Morphine injection solution (see recipe )
Novel chemical entity (NCE) solution for testing

Standard rat cages
Animal balance
Sound‐attenuating chamber equipped with an exhaust fan (MED Associates, http://www.med‐associates.com/)
Operant conditioning box (Fig. ), equipped with a balance arm located on the exterior surface of the roof and a food hopper for delivery of food pellets; the interior of the operant box is equipped with 2 retractable levers, 2 cue‐lights, a food pellet magazine (located between the two levers), a grid floor, and a house light located at the top of the opposite wall (MED Associates, cat. no. Med008‐062; http://www.med‐associates.com/)
Catheters (Model IVSA28/1350; CamCaths, http://www.camcaths.com/index.html)
50‐ml beaker
Vaporizer for isoflurane anesthesia (Single Animal Vaporizer Unit and Induction Chamber; Braintree Scientific, cat. no. EZ 17 85)
Scavenger (a pass‐through charcoal canister filter in the exhaust gas line to collect vaporized isoflurane), oxygen
Hair clippers/shaver
Surgical tools: small sharp‐tipped scissors 12 cm long with 4‐cm‐long blades; blunt‐tipped hemostatic forceps 15 cm long with 5‐cm‐long blades, two curved blunt‐tip forceps, one fine‐tipped forceps (tip: 0.05 × 0.01 mm), scalpel, and blades (#10)
Sterile gauze
Hemostats
Syringe and flusher (blunted 22‐G needle tip with attached 8‐ to 15‐cm length of Tygon tubing)
Syringe (3‐ to 5‐ml) and needle for irrigation (18‐G for high flow)
1‐ml syringes and 25‐G needles for carprofen administration (for s.c. injection)
Wound stapler and wound clips
Cotton swabs (Q‐tips)
Sutures (silk and chromic gut)
Plastic tubing (Tygon tubing is an excellent option in terms of flexibility and durability: 0.05 mm internal diameter, 0.5 mm outer diameter)
Syringe pump (MED Associates, cat. no. PHM‐111‐EC; http://www.med‐associates.com/)
Blunted 22‐G needle tips for attachment of tubing to syringe.
Syringe: 10 ml (ensure that syringe pump is calibrated correctly for syringe size)
Single‐channel fluid swivel (Fig. ) mounted on balance arm located on the roof of the operant conditioning box
A steel spring sleeve to sheath the Tygon tubing inside the operant box, with a threaded connector to attach to the catheter exit port [PlasticsOne (http://www.plastics1.com/PCR/Preclinical‐Research‐Components.php) manufactures a suitable connector, which they will provide at the specified length (Model C313CS/SP]; the length depends on the internal dimensions of the operant box: ideally, there is sufficient tubing and spring to allow connection with the catheter exit port on the rat's back (mid‐scapulae) at all times during the test session; excessive extra length tends to distract animals or get tangled as the animal moves around the box
Data acquisition and analysis software (MED PC, MED Associates; http://www.med‐associates.com/)
Computer and interface to allow control of apparatus and software, and collection and storage of data (MED‐SYST‐8 for 8 operant chambers or MED‐SYST‐16 for 16 operant chambers, MED Associates; http://www.med‐associates.com/)

Additional reagents and equipment for parenteral injection of the rat (Donovan and Brown, )

Alternate Protocol 1: Self‐Administration in Drug‐Naïve Rats

Materials

Rats: male Wistar or Sprague‐Dawley rats, 300 to 324 g upon arrival
Standard rat chow
Standard rat bedding
Dustless purified precision pellets (45 mg) for food‐shaping (Bio‐Serv, cat. no. F0165; http://www.bio‐serv.com/)
Training drugs:
- Nicotine solution (for protocol 3 ; see recipe )
- Cocaine solution (for protocol 3 ; see recipe )
70% ethanol
Novel chemical entity (NCE) solution for testing

Standard rat cages
Animal balance
Sound‐attenuating chamber equipped with an exhaust fan
Operant conditioning box (Fig. ), equipped with a balance arm located on the exterior surface of the roof, and a food hopper for delivery of food pellets; the interior of the operant box is equipped with 2 retractable levers, 2 cue‐lights, a food pellet magazine (located between the two levers), a grid floor, and a house light located at the top of the opposite wall
Data acquisition and analysis software (MED PC, MED Associates; http://www.med‐associates.com/)
Computer and interface to allow control of apparatus and software, and collection and storage of data

Basic Protocol 2: Drug Discrimination in Rats

Materials

10‐ to 12‐week‐old C57Bl6/J mice (male mice are preferable because there is no requirement to consider estrus cycle as a potential factor in the effects of the test compounds)
Standard lab mouse chow
Cleaning agent: e.g., Nolvasan (Fort Dodge Animal Health)
Test solutions dissolved in saline or sterile water, and administered in a volume of 10 ml/kg.

Light meter
OF (open‐field) test chamber: approximately 25 cm (l) × 25 cm (w) × 20 cm (h) with infra‐red (I/R) array (Med‐OFA‐MS; MED Associates, http://www.med‐associates.com/)
Animal balance
Data acquisition and analysis software (MED PC, MED Associates; http://www.med‐associates.com/)
Computer

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Figures

Figure 5.62.1 Rat operant box. The photograph shows a rat operant box, as described in the text. It should be noted that the syringe pump is placed on top of the sound‐attenuating chamber during testing, but was placed on top of the operant box itself for the photograph. In the picture, the tubing and metal sheath are situated to the side of the operant box, leading from the swivel that can be seen at the very top of the inside of the sound‐attenuating chamber. The cue lights and the housing for the retractable levers can be seen situated on the exterior of the lower right wall of the operant box. The food hopper is clearly visible, connected to the magazine by a wide plastic tube, and with a manual over‐ride button to allow testing of pellet delivery.

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Figure 5.62.2 Fluid swivel. The photograph shows a picture of an Instech fluid swivel (Model 375/22; Instech Laboratories, Inc.), used to allow delivery of the drug solution from the loaded syringe pump to the catheter port.

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Figure 5.62.3 Intravenous catheterization of the external jugular vein in the rat. Panel (A ) shows the dorsum of the rat, shaved and prepared for surgery, with a black mark identifying the target for the catheter exit port. Panel (B ) shows an isolated external jugular vein, lying across the end of a Q‐tip, ready for placement of the suture threads and the insertion of the catheter. Panel (C ) shows the catheter located in the external jugular vein, fixed in place with sutures located above and below the silicon bead. Panel (D ) shows the end of the surgical procedure, with the catheter exit port connected to a syringe filled with heparinized saline. The wound has been closed with wound clips; Bacitracin will be applied before the syringe is disconnected, the catheter plug inserted, and the rat returned to a recovery cage.

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Figure 5.62.4 Cocaine (A ) and morphine (B ) self‐administration in Sprague‐Dawley rats. Panel A illustrates how rats adjust their lever‐pressing behavior as the fixed‐ratio requirement is increased over days, in order to maintain stable levels of drug intake. The unit dose of cocaine was 0.3 mg/kg/infusion, and the time‐out was 20 sec. The rats were approximately 4 months old. Panel B illustrates how a variation in dose results in different levels of intake. Each dose was made available for five consecutive test sessions. Morphine was available on a fixed‐ratio 3 time‐out 20‐sec (FR3TO20s) schedule. In both studies, test sessions lasted 1 hr. Statistical method: Student Newman‐Keuls post‐hoc tests, following significant main effect of dose in a repeated‐measure ANOVA. Data are expressed as mean ± SEM. Asterisks ~undefined p < 0.05, ** p < 0.01) indicate significant differences compared to vehicle.

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Figure 5.62.5 Nicotine discrimination in Sprague‐Dawley rats. Panels (A ) and (B ) depict the acquisition of the nicotine (0.4 mg/kg, i.p.) DS in 3‐ to 4‐month‐old Sprague‐Dawley rats ( n = 18). Open circles represent saline‐pretreatment sessions, and closed circles nicotine‐pretreatment sessions. Graphs (C ) and (D ) show the DS properties of nicotine and varenicline, respectively, in nicotine‐trained rats ( n = 9) at 18 months of age. The rats were previously exposed to a range of nicotinic agents. Data are expressed as mean ± SEM. Dotted lines indicate the thresholds for full (80%) versus absent (20%) generalization.

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Figure 5.62.6 Locomotor sensitization induced by exposure to cocaine or D ‐amphetamine in C57Bl6/J mice. Panels (A ) and (C ) show total locomotor activity, measured daily during baseline (3 days of vehicle) or LS induction (5 days of vehicle, cocaine, or D ‐amphetamine) phases ( n = 10/group). Panels (B ) and (D ) show total locomotor activity measured after administration of a challenge dose of cocaine (10 mg/kg, i.p.) or D ‐amphetamine (1.5 mg/kg, i.p.) following the 10‐day wash‐out period. Statistical method: Student Newman‐Keuls post‐hoc tests, following significant interaction effects of Challenge X Exposure in a repeated‐measure ANOVA. Asterisks ~undefined p < 0.05) indicate a significant difference compared to either Day 1 of drug exposure (panels A and C) or the repeated vehicle‐acute challenge groups (panels B and D).

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Videos

Literature Cited

Literature Cited
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Assessment of Substance Abuse Liability in Rodents: Self‐Administration, Drug Discrimination, and Locomotor Sensitization

Abstract

Table of Contents

Materials

Basic Protocol 1: Drug Self‐Administration in Rats

Alternate Protocol 1: Self‐Administration in Drug‐Naïve Rats

Basic Protocol 2: Drug Discrimination in Rats

Figures

Videos

Literature Cited