Benzoyl chloride

All chemicals, drugs and reagents were purchased from Sigma Aldrich (St. Louis, MO) unless otherwise noted. Water and acetonitrile are Burdick & Jackson HPLC grade purchased from VWR (Radnor, PA). 10 mM stock solution of each analyte was made in HPLC water and kept at −80 °C. Standard mixture was diluted from stock using aCSF consisting of 145 mM NaCl, 2.68 mM KCl, 1.10 mM MgSO₄, 1.22 mM CaCl₂, 0.50 mM NaH₂PO₄, and 1.55 mM Na₂HPO₄, adjusted pH to 7.4 with 0.1 M NaOH. Calibration curves were made using standards at 50 nM, 500 nM, 1000 nM, 5000 nM, 10000 nM for Gly, Ser and Tau; 5 nM, 50 nM, 100 nM, 500 nM, 1000 nM for Asp, Glu, GABA, Hist, Ado, HVA, 5-HIAA and DOPAC; 0.5 nM, 5 nM, 10 nM, 50 nM, 100 nM for ACh, 5-HT, NE, DA, NM and 3-MT. Internal standard was 1 mM Gly, Ser and Tau, 100 µM Asp, Glu, GABA, Hist, Ado, HVA, 5-HIAA, and DOPAC and 10 µM 5-HT, NE, DA, NM and 3-MT derivatized with ¹³C₆ benzoyl chloride using the same procedure as ¹²C reagent, then diluted 100 fold in DMSO containing 1% formic acid. d4-ACh (C/D/N isotopes, Pointe-Claire, Canada) was spiked into the reaction mixture to a final concentration of 100 nM.

Adult male Sprague-Dawley rats (Harlan, Indianapolis, IN) weighing between 295 and 355 g were used. Ketamine (65 mg/kg i.p.) and dexdormitor (0.25 mg/kg i.p.) were used for operative anesthesia. For dual probe experiments, concentric microdialysis probes (250 µm diameter) were implanted unilaterally in both the VTA (1 mm long probe) and NAc (1.5 mm long probe) according to following coordinates from bregma and dura: anterior-posterior (AP) −5.3, medial-lateral (ML) ± 0.5, dorsal-ventral (DV) − 8.0 mm and AP + 1.2, ML ± 1.4 and DV − 7.8 mm, respectively. For single probe experiments, probes (3 mm long) were implanted into the mPFC from bregma and dura: AP +3.0, ML ± 0.5, DV − 4.0 (Paxinos and Watson 2007). Probes were secured to the skull by acrylic dental cement and metallic screws. Following surgery, rats were allowed to recover and experiments were performed later. Animals were awake and freely moving with access to food and water throughout the experiment. Microdialysis probes were flushed at 1.5 µL/min with aCSF for 3 h using a Chemyx (Stafford, TX) Fusion 400 syringe pump. Perfusion flow rate was reduced to 0.6 µL/min and samples were collected every 20 min for VTA-NAc experiments. For mPFC experiments, perfusion flow rates were reduced to 1 µL/min to generate 1 µL samples. 1 µL fractions were diluted with 4 µL aCSF, then treated same way as 5 µL sample described below. Following collection of basal fractions, VTA lines were switched to aCSF containing 50 µM bicuculline for the duration (2 h) of experiments. Though 12 µL of dialysate were collected per fraction, only 5 µL of total volume was used for analysis. For mPFC, following 20 min of basal fraction collections, 1 µM neostigmine was perfused through the probe for 5 min.