In vivo microdialysis

Freely moving microdialysis was used to determine how extracellular GABA in the interstitial fluid varies in adults versus adolescents. Microdialysis guide cannulae (BASinc, West Lafayette, IN) were stereotaxically implanted and counter-balanced in a separate cohort of adult and adolescent rats above the NAc. Adult (P70-90) coordinates were anteroposterior +1.2 mm, lateral ±2 mm, and ventral –5.2 mm. Coordinates are relative to bregma, midline, and skull surface, respectively. Adolescent (P28-35) coordinates were calculated individually using a bregma/lambda ratio in comparison with adult bregma/lambda measurements (~9 mm adult distance). For example, if the distance between bregma and lambda was 8.3 mm for a single adolescent, our ratio for that individual would be 8.3 mm/9 mm. The result (i.e. 0.92) would then be multiplied by adult anteroposterior, lateral, and ventral coordinates to calculate new adolescent coordinates. Adolescent coordinates exhibited slight individual variation. All rats were allowed 3–5 days recovery time following surgery before the start of any experimentation. Microdialysis probes (1 mm membrane length; BASinc) were inserted ~1 hr before the first sample collection. Tissue was continuously perfused at 1 µL/min throughout the duration of the experiment with artificial cerebrospinal fluid aCSF (pH 7.4) containing (in mM): NaCl (148), KCl (2.7), CaCl2 (1.2), MgCl2 (0.85). Collections occurred 3 hr into the dark cycle across 6 hr with samples collected every 20 min (i.e. 3 samples per hour). During the first 3 hr, 9 samples were collected as baseline samples. Following baseline collections, α-Ctx (1.6 µL) was then infused at 0.2 µL/min over 8 min. During the 3 hr post α-Ctx, nine additional samples were collected starting 10 min following the start of α-Ctx infusion in 20 min intervals. Upon completion of microdialysis sampling, rats were euthanized and brain extracted. Slices containing the NAc were then used to confirm proper placement of microdialysis probe by recording live tissue probe tracts in all rats.