Operant conditioning maintained by cocaine
Operant self-administration apparatus. The self-administration experiments were conducted in mouse operant chambers (Model ENV-307A-CT; Med Associates Inc., Georgia, VT, USA) equipped with two holes, one randomly selected as the active hole and the other as the inactive hole as previously described (Gutierrez-Cuesta et al., 2014).
Operant conditioning maintained by cocaine. Cocaine self-administration experiments were performed as previously described (Gutierrez-Cuesta et al., 2014; Martín-García et al., 2015a). Mice were deeply anesthetized by intraperitoneal injection of a mixture of ketamine (75mg/kg; Imalgène; Merial Laboratorios S.A., Barcelona, Spain) and medetomidine (1mg/kg; Domtor; Esteve, Barcelona, Spain). After surgery, anesthesia was reversed by subcutaneous injection of the synthetic α2 adrenergic receptor antagonist, atipamezole (2.5mg/kg; Revertor; Virbac, Barcelona, Spain) indicated for the reversal of the sedative and analgesic effects of medetomidine (α2 adrenergic receptor agonist). In addition, mice received an intraperitoneal injection of gentamicin (1 mg/kg; Genta-Gobens; Laboratorios Normon, S.A., Madrid, Spain) along with subcutaneous administration of the analgesic meloxicam (2 mg/kg; Metacam; Boehringer Ingelheim, Rhein, Germany). Each 2h daily self-administration session started with a priming injection of the drug. Cocaine (obtained from The Spanish Agency of Drugs, Madrid, Spain) was intravenously infused in 23.5μl over 2s (0.5 mg/kg per injection). Cue light, located above the active hole, was paired with the delivery of the reinforcer. Mice (WT n=36; Plcb1+/- n=26) were trained under a fixed ratio 1 schedule of reinforcement (FR1; one nose-poke lead to the delivery of one dose of cocaine) over 5 consecutive daily sessions and under a fixed ratio 3 (FR3) over 5 consecutive daily sessions. Control mice trained with saline were included for both genotypes (WT n=6; Plcb1+/- n=6). The timeout period after infusion delivery was 10 s. Responses on the inactive hole and all responses elicited during the 10-s timeout period were also recorded. Responses during the 10-s timeout period were considered a measure of impulsivity reflecting the inability to stop motor behavior once it is initiated. The criteria for self-administration behavior were achieved when all of the following conditions were met: (1) mice maintained stable, responding with 20% deviation from the mean of the total number of reinforcers earned in three consecutive sessions (80% of stability); (2) at least 75% of mice responding on the active hole; and (3) a minimum of 10 reinforcers per session.
After the 10 FR sessions, animals were tested in a progressive ratio schedule of 4h where the response requirement to earn the cocaine escalated according to the following series: 1–2–3–5–12–18–27–40–60–90–135–200–300–450–675–1000. Then, we proceed with the extinction phase, 2h daily sessions following the same experimental conditions than the cocaine self-administration sessions except that cocaine were not available, and cue-light was not presented. Mice were given 2-h daily sessions until they achieved the extinction criterion with a maximum of 26 sessions. The criterion for extinction was achieved when, during 3 consecutive sessions, mice completed a mean number of nose pokes in the active hole consisting of 30% of the mean responses obtained during the 3 days to achieve the acquisition criteria for cocaine self-administration training. On day 27, only mice that accomplished extinction criterion were tested in the cue-induced reinstatement during a 2h session, to evaluate the reinstatement of cocaine-seeking behavior. The test for cue-induced reinstatement was conducted under the same conditions used in the training phase except that cocaine was not available. The reinstatement criterion was achieved when nose pokes in the active hole were double the number of nose pokes in the active hole during the 3 consecutive days when the mice acquired the extinction criteria.
The catheter was flushed daily with heparinized saline (30 USP units/ml). The patency of intravenous catheters was evaluated after the last cocaine self-administration session and whenever the behavior appeared to deviate dramatically from that observed previously by infusion of thiopental through the catheter. If prominent signs of anesthesia were not apparent within 3s of the infusion, the mouse was removed from the experiment. The success rate for maintaining patency of the catheter (mean duration of 11 days) until the end of the cocaine self-administration training was 90%.
After the cue-induced reinstatement, animals were euthanized by decapitation, brains were quickly removed, and the medial prefrontal cortex (mPFC) and hippocampus (HPC) were dissected. Brain tissues were then frozen by immersion in 2‐methylbutane surrounded by dry ice and stored at −80°C for later RNA isolation and transcriptomic analyses.