Cma 7

For in vivo microdialysis experiments to examine behavioral plasticity, cannulae for were targeted to RA bilaterally with stereotaxic coordinates as described previously^{53 (link)}, and phosphate-buffered saline or the mGlur2/3 antagonist LY341495 (100–200 nM) was infused into RA through microdialysis probes (CMA-7, Harvard Apparatus). After the final experiment, birds were infused with fluorescent muscimol-BODIPY (Life Technologies, 1 mM) to confirm targeting of the cannulae and drug spread to RA.

Mice were implanted with a dual cannulae system (Plastics One, Roanoke, VA) to bilaterally target the mouse posterior VTA (AP-3.2, ML±0.75, DV-4.5mm from bregma and dura). Dummy cannulae and dust caps fitted the length of the cannula while VTA dual injectors protruded 0.1mm past the cannula. Other groups of mice were implanted with a unilateral VTA cannula and an ipsilateral microdialysis guide cannula (CMA7, Harvard Apparatus, Holliston, MA) targeting the NAc shell (AP+1.7, ML-0.7, DV-4.0mm from bregma and dura). On test days, doses of the CRF-R1 antagonist CP376395 (0.3–0.6µg, Tocris, Ellisville, MO) or CRF-R2 antagonist astressin2B (0.25–0.5µg, Tocris, Ellisville, MO) were freshly dissolved in an artificial cerebral spinal fluid (aCSF) vehicle (0.2µl infused at 0.1µl/min). Doses were chosen based on previous microinjection procedures (Hwa et al. 2013 (link), Boyson et al. 2014 (link)). EtOH and H2O were given to animals 10 min post-infusion. After testing, mice were intracardiacally perfused with 0.9% saline and 4% paraformaldehyde, followed by brain removal. Coronal sections were Nissl stained to check placement of guide cannulae. Animals with incorrect placements into target sites were excluded from analysis.

The night before sample collection, the microdialysis probe with a 1-mm active membrane (CMA7, Harvard Apparatus, Holliston, MA) was inserted into the NAc under isoflurane anesthesia (Webster Veterinary, Devens, MA) at an overnight perfusion rate of aCSF at 0.5 µl/min. On the test day, the flow rate was increased to 1.5 µl/min for 1 hr until sample collection every 10 min. There were four baseline samples followed by 0.2 µl microinjections of aCSF and 0.6 µg CP376395. Microinjection of the CRF-R1 antagonist occurred 10 min before 3 hr into the dark cycle, mimicking microinjection procedures when two-bottle choice was given (Hwa et al. 2014 ). Dopamine was measured by HPLC by electrochemical detection (Miczek et al. 2011 (link)). A stabilizing agent of 20 mM phosphate buffer with 25 mM EDTA was added to 15 µl dialysate samples. A cation-exchange column (Capcell Pak SCX, 1.5mm × 250mm, 5 µm I.D, Shiseido, Tokyo, Japan) separated dopamine at 30°C and a flow rate of 0.2 ml/min. The mobile phase consisted of 150 mM ammonium acetate, 50 mM citric acid, 27 µM EDTA, 10% MeOH, and 1% acetonitrile with pH adjusted to 4.6. Dopamine concentrations were determined by using standard curves with known amounts of dopamine in a range of 0.125–0.5 pg. The limit of detection was 2 fg under these conditions with a 10.5% recovery rate.