Microdialysis and HPLC

Three separate batches of animals were used for the microdialysis experiments (Batch 1, n=2; Batch 2, n=4; Batch 3, n=4). Beginning three days after cannula implantation, animals were habituated in microdialysis bowls for 3–4 days and habituated with intranasal saline for 7 days. On microdialysis days, guide cannula stylets were removed and substituted with a microdialysis probe (BASi, West Lafayette, IN, USA) that extended 2 mm past the guide cannula. Probes were perfused at a 2 µl/min flow rate with artificial cerebrospinal fluid (aCSF, pH 7.4) containing: 150 mM NaCl, 3mM KCl, 1.7mM CaCl₂, 0.9 mM MgCl₂, and 4.9 mM _D-glucose. Neostigmine bromide (50 nM; Sigma) was added to the aCSF to increase recovery of acetylcholine in collected dialysates. Dialysate collection started after a 3 hour discard period. Microdialysis sessions consisted of one hour (4 × 15 minute collections) of baseline collections followed by intranasal vehicle (0.9% saline) or OxA (100 uM; Enzo Life Sciences), administered in a total volume of 50 µl in 12.5 µl increments over a 2-minute period. The individual performing intranasal administration was not blinded to treatment condition. Dialysate collection then continued for two hours (8 × 15 minute collection) post-treatment. Upon collection, dialysates were stored at −80°C until analysis. All animals underwent two separate microdialysis sessions with an off day in-between and experiments were counterbalanced so that half of the animals received vehicle during session one, while the other half received OxA in session one. The day following the last microdialysis session, rats were euthanized and their brains processed for probe placement verification using an acetylcholinesterase (AChE) background stain. Probe placement verification was performed after all HPLC samples were run for each respective animal. Any probe placement visualized outside the medial PFC excluded the animal from the study.

Each 30 µl dialysate was split prior to analysis by high performance liquid chromatography with electrochemical detection (HPLC-ECD), with 20 µl analyzed for ACh and 10 µl analyzed for glutamate. ACh was analyzed using an HTEC-510 HPLC-ECD (EicomUSA; San Diego, CA, USA). Briefly, 20 µl of each dialysate was loaded into the AC-GEL separation column (2.0 ID × 150mm; EicomUSA) maintained at a constant 33°C in combination with mobile phase (pH 8.5) containing 49.4 mM potassium bicarbonate (KHCO₃), 134.3 µM ethylenediaminetetraacetic acid disodium (EDTA-2Na), and 1.23 mM sodium 1-decanesulfonate. After analyte separation, post-column derivatization of ACh was attained through use of an AC-ENYM II enzyme reactor (1.0 ID × 4 mm; EicomUSA) containing acetylcholinesterase and choline oxidase, which generates hydrogen peroxide (H₂O₂₎ proportional to the amount of ACh present. The H₂O₂ is further broken down and detected on a platinum working electrode with an applied potential of +450 mV. The amount of ACh in each sample was measured by comparison with a three-point external standard curve with values predicted to be in range of the collected dialysates. The limit of detection for this analysis was approximately 5 fmol/injection

Glutamate was analyzed using a CC-32 HPLC-ECD (BASi; West Lafayette, IN, USA) with modifications. First, 10 µl of each dialysate was loaded into the GU-GEL separation column (4.6 ID × 150mm; EicomUSA) in conjunction with a mobile phase (pH 7.2) containing 60 mM ammonium chloride-ammonium hydroxide, 134.3 nM EDTA-2Na, and 686 µM hexadecyltrimethylammonium bromide. After separation, post-column derivatization of glutamate was attained through the use of an E-ENZ enzyme reactor (3.0 ID × 40 mm; EicomUSA) containing glutamate oxidase, which generates H₂O₂ proportional to the amount of glutamate present. The H₂O₂ is further broken down and detected on a 3.0 mm glassy carbon electrode (BASi) coated with a horseradish peroxidase osmium polyvinylpyridine solution (0 mV applied potential). The amount of glutamate in each dialysate was measured by comparison with a three-point standard curve using external standards expected to be in range of the collected dialysates. The limit of detection for this method was approximately 3 fmol/injection.