In Your Lab:
Intravenous Self-Administration of Drugs of Abuse in Mice
Article submitted by: Robyn Brown and Andrew J Lawrence, Florey Neuroscience Institutes and Centre for Neuroscience Research, University of Melbourne, Parkville, Victoria, Australia
Robyn Brown and Andrew Lawrence in the rodent behavioral facility at the Florey Neuroscience Institutes located on the campus of the University of Melbourne, Parkville, Australia.
Operant self-administration is a procedure commonly used to measure drug reinforcement and drug-seeking behavior in animals. The increasing popularity of the use of transgenic mice in biomedical research has led to a need for establishing a reliable protocol for chronic intravenous self-administration which is more suited to this small rodent. Our laboratory has established a protocol which enables the assessment of a number of addiction-relevant behaviors in this species.
In our laboratory, operant self-administration of either oral sucrose/ethanol or intravenous drugs is assessed using operant chambers (model ENV-307W, Med Associates, Vermont, USA) which are equipped with two levers, one paired with reward delivery (the active lever) and one which results in no outcome when pressed (the inactive lever, see figure 1). A stimulus light (conditioned stimulus, CS) is located above the active lever and flashes on for a period of 10 s when the active lever is pressed and drug/sucrose delivered. In addition to this visual cue, a tray is placed under the grid below the active lever with a piece of paper (1 centimeter (cm) x 1 cm) upon which two drops of vanilla essence are placed before each session. The vanilla infused paper provides an olfactory cue (discriminative stimulus) for the location of the active lever and indicates drug availability. The chambers are housed in sound attenuated boxes and ventilated with fans. Note, a similar set-up could be employed with nose-pokes as opposed to levers.
Figure 1: A schematic of the operant chamber.
Operant conditioning experiments are conducted in reverse light cycle conditions (dark phase 7am-7pm) so as to enable experimentation during the most active period for the mice. Unlike rats, mice may benefit from training with a food reward before intravenous drug to facilitate acquisition of the instrumental task. This issue really depends upon the strain of mouse used and it is possible for mice to acquire drug self-administration without the assistance of food training. In addition, as our laboratory commonly assesses mice which have had a specific gene mutated or deleted, assessing self-administration of a natural reward such as sucrose provides a valuable control to ascertain whether the genetic manipulation affects instrumental learning more generally, which could confound interpretation of results. Clearly however, if the specific goal of the study is to assess acquisition of drug self-administration then prior food training should be avoided.
Implantation of the venous cannula is a relatively quick surgery (between 10-20min), thus a light inhalation anaesthetic is ideal (we use isoflurane at 1.5-2%). A stereoscope enables viewing of the jugular vein insertion point. Our cannula are home-made and consist of a 3.5 cm length of silastic® tubing (ID 0.012 inches x OD 0.025 inches) attached to a 22 gauge needle which is bent into a U shape, then bent again at right angles to the luer. The catheter is inserted 1 cm into the jugular vein and anchored with suture. The remaining tubing runs subcutaneously behind the ear to exit on top of the head. The catheter port is attached to the skull using adhesive followed by dental cement. Whilst still under anesthesia the catheter is flushed with heparinised antibiotic and mice receive Meloxicam (1-3 mg/kg ip) for post-operative analgesia. After surgery mice are allowed to recover (up to 3-4 days) before experiments begin. Catheter patency is maintained by twice daily flushing with heparinised saline (typically before and after a self-administration session). The patency of the catheters is evaluated using 0.02-0.03 ml of a ketamine (15 mg/ml) and midazolam (0.75 mg/ml) solution. If prominent signs of hypnosis are not apparent within 3 s of infusion mice should be removed from the experiment. Other reassuring signs of catheter patency include observable changes in levels of responding with changing dose of drug or fixed ratio requirement and stable lever discrimination.
When running self-administration sessions mice are connected via the jugular catheter to an intravenous line (Tygon®, ID 0.02 inches, OD 0.06 inches) connected to a 22 gauge swivel (Instech Solomon). The swivel is connected to a syringe held in an infusion pump (model PHM-100VS) with BCoex®-T22 tubing (ID 0.6 mm x OD 1.6 mm). A fixed ratio of 1 (FR1) active lever press per drug delivery is typically employed. Intra-venous drug sessions are typically 2 h in length and held at approximately the same time each day. A maximum is set to prevent overdose as well as a 10 s time out period after each drug infusion. During the time out period active lever presses are recorded but no drug infusion occurs. Criteria are set and these are usually specific to the reinforcer being assessed e.g. see Brown et al., 2009 for morphine, McPherson et al., 2010 for cocaine and Cahir et al., 2011 for nicotine. Mice that do not reach criteria should be excluded from the study. Care should be taken to ensure that application of criteria does not inadvertently prevent a phenotype from being observed e.g. deletion of a particular gene prevents self-administration at a level which satisfies criteria thus all mutant mice are excluded from the study.
Motivation can be measured by assessing ‘breakpoint’ on a progressive ratio (PR) schedule. Breakpoint represents the point at which an animal ceases to press the active lever for a drug infusion when the instrumental requirement is progressively increased. This reflects the motivation of an animal to self-administer a given drug and may therefore provide more accurate data pertaining to the reinforcing efficacy of that drug (Arnold et al., 1997). The PR protocol utilized by our lab is modified from Thompson et al., (2005). The breakpoint is defined as the last completed ratio, after which a period of 60 min ensued where no reinforcer was earned. If this does not occur, the session is terminated after 2 h and the breakpoint is defined as the final ratio completed within the 2 h session. After mice have self-administered drug daily for approximately 2 weeks they are placed into forced abstinence in their home cage, or undergo extinction training. After extinction criteria is met (30% of FR1 responding), or following 3 weeks of forced abstinence, drug-seeking can be precipitated by placing mice into the operant chambers with the visual and olfactory cues present. Drug-seeking is assessed under extinction conditions (FR1 response results in presentation of CS but no infusion of drug) for 1 h. There is no maximum set as there is no risk of overdose. Refer to figure 2 for a schematic of this operant self-administration protocol.
Figure 2: Protocol for operant self-administration.
Our laboratory has obtained some important findings via the assessment of intravenous self-administration in mice. We have found a key role for the adenosine A2A receptor in the centrally mediated effects of morphine (Brown et al., 2009) and that deletion of CREB1 from the dorsal telencephalon reduces motivational properties of cocaine (McPherson et al., 2010). Examples of the findings regarding the adenosine A2A receptor are shown in figure 3. Deletion of the adenosine A2A receptor was found to result in decreased self-administration of morphine as well as a decreased breakpoint on a progressive ratio schedule. Our laboratory will continue to utilize this intravenous self-administration model to assess the impact of genetic manipulations on aspects of reinforcement, drug-seeking and motivated behavior. In addition, we have been able to assess the impact of pharmacological manipulations by using a within subjects design whereby mice are injected with vehicle before their operant session until responding is stable. The following session mice are injected with drug of choice (e.g. a receptor antagonist as shown in figure 4, see also Cahir et al., 2011 and Brown et al., 2012). When assessing the impact of a particular drug on drug-seeking a between subjects design is typically employed whereby half the mice are administered vehicle and half the mice are administered drug before the drug-seeking session (e.g. see Brown et al., 2012).
Figure 3. Morphine self-administration (0.1 mg/kg/infusion) on fixed and progressive ratios in A2A knockout (KO) and wildtype (WT) mice. Data are expressed as mean (± SEM), n=17-20 per genotype. (A) Average of the total number of lever presses (on the active and inactive lever) over the 7 days of self-administration; ### p<.001 compared to active lever, *** p<.001 compared to wildtype (two-way ANOVA). (B) Number of drug infusions and dose of morphine obtained over 7 days in mg/kg; *** p<.0001, **p<.001, *p<.05 significant effect of genotype (two-way ANOVA with SNK post tests). (C) Breakpoint; * p<.001 (unpaired t test). (D) Cumulative response record for the PR session divided into 10 min time bins; * p<.05 compared to wildtype for that time bin (two-way ANOVA with Holm-Sidak post tests).
Figure 4. The effect of MTEP on morphine self-administration (0.1mg/kg/infusion) in mice. Data are expressed as mean (±SEM), n = 9 per group. (A) Average of the total number of lever presses (on the active and inactive lever) over the 5 days of self-administration; ### p < 0.001 compared to vehicle, *** p < 0.001 compared to active lever (two-way ANOVA with SNK post hoc comparisons). (B) Time course of morphine self-administration over 5 days showing effect of MTEP administration on day 4; *** p < 0.001 compared to other days, ### p < 0.001 compared to inactive lever (one-way ANOVA with Tukey post hoc comparisons).
Arnold JM, Roberts DC (1997). A critique of fixed and progressive ratio schedules used to examine the neural substrates of drug reinforcement. Pharmacol Biochem Behav 57(3): 441-447.
Brown RM, Short JL, Cowen MS, Ledent C, Lawrence AJ (2009). A differential role for the adenosine A2A receptor in opiate reinforcement vs opiate-seeking behavior. Neuropsychopharmacology 34(4): 844-856.
Brown RM, Stagnitti MR, Duncan JR, Lawrence AJ (2012). The mGlu5 receptor antagonist MTEP attenuates opiate self-administration and cue-induced opiate-seeking behavior. in mice. Drug Alcohol Depend., in press.
Cahir E, Pillidge K, Drago J, Lawrence AJ (2011). The necessity of alpha4* nicotinic receptors in nicotine-driven behaviors: dissociation between reinforcing and motor effects of nicotine. Neuropsychopharmacology 36(7): 1505-1517.
McPherson CS, Mantamadiotis T, Tan SS, Lawrence AJ (2010). Deletion of CREB1 from the dorsal telencephalon reduces motivational properties of cocaine. Cereb Cortex 20(4): 941-952.
Thomsen M, Woldbye DP, Wortwein G, Fink-Jensen A, Wess J, Caine SB (2005). Reduced cocaine self-administration in muscarinic M5 acetylcholine receptor-deficient mice. J Neurosci 25(36): 8141-8149.
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