Mouse operant chamber

All tests took place in mouse operant chambers (17.8 cm × 15.2 cm × 18.4 cm; Med Associates). A rotating optical commutator (Doric) was located on the top of the operant chamber and connected to a 473 nm diode-pumped solid-state laser (OEM Laser Systems; see Fig. 2f). Fibers were connected to the implants on the mouse for every training session. The conditioning chamber light was turned on during every training session. Laser power was adjusted to obtain ~10 mW transmittance into the brain. Mice were trained to nose poke in a MED Associates operant box with two nose-poke portals available, “active” and “inactive.” Successful nose pokes on the active nose poker rewarded the mouse with 2s of stimulation (40Hz, 10ms pulse width). An orange cue light turned on above the sipper during the 2s stimulation period. Nose poking to the inactive portal produced no consequences or rewards. Once discrimination was acquired during 1 h long FR1 sessions (less than 30% of total poking in the inactive hole), mice underwent 1 session of FR3. Mice’s motivation was then assessed using a 1 h session of progressive ratio schedule of reinforcement. The data were calculated as total number of nose pokes at the active and inactive for each day over the course of the experiment^{17 (link),64 (link)}.

All tests took place in mouse operant chambers (17.8 cm × 15.2 cm × 18.4 cm; Med Associates). A rotating optical commutator (Doric) was located on the top of the operant chamber and connected to a 625 nm diode-pumped solid-state laser (For JAWS experiments) or 473 nm diode-pumped solid-state laser (OEM Laser Systems; see Fig. 2f). Fibers were connected to the implants on the mouse for every training session. Sucrose pellets were delivered at a variable ITI (VI-90s) and animal head entries were recorded by outfitting the pellet receptacles with custom-made IR sensors to record beam breaks during head entries. Upon training, animals underwent counter-balanced of laser on vs. off sessions, where laser was delivered for 10s flanking reward delivery. Laser power was adjusted to obtain ~5 mW transmittance into the brain for JAWS experiments and ~10 mW transmittance for ChR2 experiments. For the JAWS experiments, we used previously employed parameters for laser delivery – 2s on, 2s ramp down and 2s off. For ChR2 experiments, we used 20 Hz, 5ms pulse-width for laser delivery.

The self-administration sessions were conducted in mouse operant chambers (Med Associates, St. Albans, VT, USA). The chambers (21.4 cm length × 20.3 cm width × 12.7 cm height) were located in a dark room and individually enclosed in wooden sound-attenuating cubicles that were fitted with a ventilation fan that additionally masked external noise. The operant chamber consisted of clear polycarbonate side walls and ceiling, with a modular aluminum front and back walls that allowed operant manipulanda to be mounted. The floor consisted of twenty-four 3.2 mm diameter steel rods that were spaced 8.9 mm apart. Two illuminated nosepoke holes (1.3 cm diameter × 1 cm depth) that were equipped with an infrared beam were mounted 1.5 cm above the floor on the front wall. A spring-covered Tygon tube was connected to the mouse’s catheter through a fluid swivel, and the swivel was connected to a syringe that contained the heroin solution that was placed outside the chambers. The syringe was placed inside a syringe pump (Med Associates, St. Albans, VT, USA; 3.3 revolutions per minute) that was placed on top of the wooden cubicle. MedPC software controlled the delivery of fluids and presentation of visual stimuli and recorded the behavioral data.

Optogenetic experiments were designed to determine whether Xie2-64 alters DA-dependent rewarding-seeking behaviors (Han et al., 2017 (link); Jordan et al., 2019 (link)). Briefly, AAV-EF1α-DIO-ChR2-EGFP (150 nl, ~2 × 10¹² genomes/ml, University of North Carolina Gene Therapy Center) was microinjected into the VTA of adult DAT-Cre mice (AP −3.28; ML 0.43; DV −4.41 mm relative to Bregma) under anesthesia, as we reported previously (Jordan et al., 2019b ). After 4 weeks of recovery, mice were allowed to lever press in mouse operant chambers (Med Associates Inc.) to earn a one second pulse train of laser stimulation (473 nm, 20 mW, 5 ms duration, 25 Hz), paired with one second illumination of a cue light above the active lever during daily, one hour sessions. Inactive responses were recorded but had no scheduled consequences. Once reliable responding was established for 25 Hz, mice were transitioned to a rate-frequency program in which 6 different stimulation frequencies (100, 50, 25, 10, 5, and 1 Hz) were presented in descending order for 10 minutes each. After stable oICSS baselines were established, mice were treated i.p. with vehicle, cocaine alone (2 or 10 mg/kg, 5-min. prior to oICSS sessions), Xie2-64 (10 or 20 mg/kg) or SR144528 (1, 3 mg/kg, 30-min prior to oICSS sessions), either alone or 30-min prior to cocaine (10 mg/kg) and oICSS sessions.