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.