In vivo microdialysis

Mice were anaesthetized (pentobarbital, 50 mg/kg, i.p.) and stereotaxically implanted with concentric microdialysis probes into the CA3 (AP: − 2.46 mm from bregma, L: 2.75 mm from midline and V: 2.6 mm below dura or dentate gyrus (DG, AP: − 1.7 mm from bregma, L: 1 mm from midline and V: 1.8 mm below dura) areas of the hippocampus [46]. Microdialysis concentric probes were constructed as described by Hutson et al. [47] with 25G (0.3 mm inner diameter, 0.5 mm outer diameter, A-M Systems) stainless steel tubing and inlet and outlet cannulae (0.04 mm I.D., 0.14 mm O.D.) consisting of fused silica tubing (Scientific Glass Engineering, Melbourne, Australia). The microdialysis probe had a tubular dialysis membrane (Enka AG, Wuppertal, Germany) of 0.8 mm in length. After 24 h of recovery, in vivo microdialysis was performed in awake and freely moving mice by perfusing dialysis probes with artificial cerebrospinal fluid (CSF composition in mM: KCl, 2.5; NaCl, 125; MgCl₂, 1.18; CaCl₂, 1.26) (pH 7.2) at a rate of 1.0 μl/min using a Harvard Apparatus infusion pump (mod. 22). After a 60 min equilibration period, 6 consecutive 30-min dialysate samples were collected for detecting the basal release of neurotransmitters following PEA-OXA or vehicle chronic treatments. For single PEA-OXA or vehicle administration, 12 consecutive dialysate samples were collected (5 pre-administration and 7 post-administration. On completion of experiments, mice were anaesthetized with pentobarbital and their brains perfused–fixed via the left cardiac ventricle with heparinized paraformaldehyde saline (4%). Brains were dissected out and fixed in a 10% formaldehyde solution for 2 days. Each brain was cut in 40 μm thick slices and observed under a light microscope to identify the probe tip locations.

Dialysates were analyzed for amino acid content using high-performance liquid chromatography (HPLC) with the fluorimetric detection method. The system comprised a Varian ternary pump (mod. 9010), a C18 reverse-phase column, a Varian refrigerated autoinjector (mod. 9100) and a Varian fluorimetric detector. Dialysates were precolumn derivatized with o-pthaldialdehyde-N-acetylcysteine (OPA-NAC) (10 μl dialysate + 5 μl OPA-NAC + 10 μl borate buffer 10%) and amino acid conjugates re-solved using a gradient separation. The mobile phase consisted of two components: (1) 0.2 M sodium phosphates buffer and 0.1 M citric acid (pH 5.8) and (2) 90% acetonitrile and 10% distilled water. Gradient composition was determined using an Apple microcomputer installed with Gilson gradient management software. Data were collected using a Dell Corporation PC system 310 interfaced to the detector via a Drew data-collection unit. The mean dialysate concentration of amino acids in the six samples represented the basal release and the results were expressed as the mean ± SEM of the pmol in 10 μl of perfusate sample.

Biogenic amines (norepinephrine, dopamine and histamine) content was assessed by HPLC with the electrochemical detector method, consisting of a model 590 pump (Waters Associates, Milforal, USA) and an electrochemical detector BIORAD mod. 1640 with an Ag/AgCl reference electrode. A C-18 reverse-phase analytical column (Discovery, 5 µm, 4.6 mm i.d. × 150 mm; SUPELCO, USA) was eluted using different mobile phases depending on the amine and maintained at a fixed temperature of 40 °C. Mobile phase composition and flow rate were: 12.5% methanol, 0.15 mM NaH₂PO₄, 0.01 mM octanosulfonic acid, 0.5 mM EDTA (pH 3.8), flow rate 1 ml/min for dopamine; 12% methanol, 0.1 M sodium acetate, 0.3 mM EDTA, 1.8 mM octanosulfonic acid (pH 5.4), flow rate 0.8 ml/min for norepinephrine; and 20% methanol, 100 mM NaH₂PO₄, 5.2 mM octanosulfonic acid (pH 6), flow rate 0.8 ml/min for histamine. Detection potential was set at 0.55 V. Volume injection was 10 µl for dopamine and norepinephrine and 20 µl for histamine which requires derivatization with OPA-Na₂SO₃ solution [48] in a ratio of 10:2 (10 µl of dialysate and 2 µl of derivatization solution, and 8 µl of ACSF). OPA-Na₂SO₃ stock solution was composed of OPA 25 mM, sodium sulfite 125 mM, and 0.1 mM sodium borate buffer (pH 10.4). The mean concentration of norepinephrine, dopamine and histamine in the six dialysate samples represented the basal release and the results were expressed as the mean ± S.E.M of the fmol in 10 µl of perfusate. For the single PEA-OXA or vehicle administration the percentage of concentration variation after administration was considered.