Stereotaxic surgery and probe insertion

Stereotaxic surgery was performed during the 7^th week of EtOH drinking. Rats were stereotaxically implanted ipsilaterally with two guide cannulae (Plastics One, Inc., Roanoke, VA, USA) under 2% isoflurane inhalation anesthesia for microinjection-microdialysis experiments. The 18-gauge cannulae were used for microdialysis probes, and the 22-gauge cannulae were used for microinjection (Ding et al 2009b, 2011). One cannula was aimed above the pVTA (AP −5.6 mm, ML +2.1 mm, DV −9.0 mm), and the other above the NACsh (AP +1.7 mm, ML +2.3 mm, DV −8.5 mm, Paxinos & Watson 1998). For reverse microdialysis, one 18-gauge cannula was implanted in to the pVTA. The cannulae were implanted at a 10° angle to the vertical line. The study of pVTA is based on previous findings that the pVTA is the anatomical substrate supporting the reinforcing and stimulating effects of ethanol. For example, ethanol can be self-infused into the pVTA but not anterior VTA (Rodd et al 2005b, Rodd et al 2004). In addition, microinjection of ethanol into the pVTA but not the anterior VTA increased dopamine release in the NACsh (Ding et al 2009b). Rats were allowed to recover from surgery for at least 5 days prior to the microdialysis experiment. Approximately 16–18 hr before microdialysis, a loop-style probe (active membrane length 1.5 mm for NACsh and 1.0 mm for pVTA (inner diameter 200 μm, molecular weight cut-off: 13,000, Spectrum Laboratories, Inc, Rancho Dominguez, CA, USA) was inserted into the NACsh or pVTA, as previously described (Ding et al 2009b).